Feather meal processing methods impact the production parameters, blood biochemical indices, gut function, and hepatic enzyme activity in broilers.

This study investigated the effects of feather meal (FM) processing methods on production parameters, blood biochemical indices, intestinal morphology, digestive and hepatic enzyme activities, and gastrointestinal tract pH and microflora of broilers. A total of 480-d-old male broilers were used for 42 d in a completely randomized design with eight treatments and five replicates (12 chicks/replicate). Treatments were 1) a control diet (without FM), 2) a diet containing 4% raw FM (RFM), 3) a diet containing 4% processed FM (PFM) by autoclave (Au-PFM), 4) a diet containing 4% fermented FM (FFM) by Bacillus licheniformis (Bl-FFM), 5) a diet containing 4% FFM by Bacillus subtilis (Bs-FFM), 6) a diet containing 4% FFM by Aspergillus niger (An-FFM), 7) a diet containing 4% FFM by B. licheniformis + B. subtilis + A. niger (Co-FFM), and 8) a diet containing 4% PFM by an enzyme (En-PFM). Results showed that in the FFMs the contents of ash, ether extract, total volatile nitrogen, and amino acids including Lys, Met, Thr, Trp, His, Leu, Gly, Ile, Phe, and Tyr increased (P < 0.05), while crude fiber, crude protein, and dry matter content decreased (P < 0.05). Compared with the control, the Co-FFM diet had no significant differences (P > 0.05) in total body weight gain (2,827 vs. 2,791 g/chick), total feed intake (5,018 vs. 4,991 g/chick), European production efficiency factor (375 vs. 377), European Broiler Index (371 vs. 371), and feed conversion ratio (1.77 vs. 1.78 g/g). Feeding FFM decreased (P < 0.05) serum total cholesterol (1.46-fold), triglyceride (1.61-fold), very low-density lipoprotein cholesterol (1.61-fold), and low-density lipoprotein cholesterol (2.27-fold) compared to the control. Also, FFM increased (P < 0.05) villus height (1,045 to 1,351, 661 to 854, and 523 to 620 µm), and villus height to crypt depth ratio (6.15 to 8.45, 4.55 to 7.04, and 4.27 to 5.45), in the duodenum, jejunum, and ileum, respectively, compared to the control. Compared to the control, the Co-FFM diet increased (P < 0.05) protease (34, 39, and 45 %) in the pancreas, duodenum, and jejunum, as well as amylase (73, and 97 %) activities in the duodenum, and jejunum, respectively. Diets containing FFM reduced (P < 0.05) pH in the crop, gizzard, and ileum, and decreased (P < 0.05) Escherichia coli (6.12 to 5.70) count in ileum compared to the control. The Co-FFM diet increased (P < 0.05) lactic acid bacteria count in crop (6.77 to 7.50) and ileum (6.94 to 7.73), also decreased (P < 0.05) coliforms (6.31 to 5.75) count in ileum compared to the control. In conclusion, FM fermentation, particularly Co-FFM, improves the nutritional value of FM, converting it into a decent source of dietary protein for broilers.

Fermentation represents an attractive alternative method for feather meal (FM) efficient bioconversion and its nutritional value enhancement. This study investigated the effects of FM processing methods on broilers. Experimental diets were 1) a control diet (without FM), 2) a diet containing 4% raw FM (RFM), 3) a diet containing 4% processed FM (PFM) by autoclave (Au-PFM), 4) a diet containing 4% fermented FM (FFM) by Bacillus licheniformis (Bl-FFM), 5) a diet containing 4% FFM by Bacillus subtilis (Bs-FFM), 6) a diet containing 4% FFM by Aspergillus niger (An-FFM), 7) a diet containing 4% FFM by B. licheniformis + B. subtilis + A. niger (Co-FFM), and 8) a diet containing 4% PFM by an enzyme (En-PFM). Results showed that FFMs increased the contents of ash, ether extract, total volatile nitrogen, and amino acids including Lys, Met, Thr, Trp, His, Leu, Gly, Ile, Phe, and Tyr, while decreased crude fiber, crude protein, and dry matter content. The production parameters of birds fed Co-FFM were similar to the control group. In addition, FFMs decreased serum total cholesterol (1.46-fold), triglyceride (1.61-fold), very low-density lipoprotein cholesterol (1.61-fold), and low-density lipoprotein cholesterol (2.27-fold). Furthermore, Co-FFM improved intestinal morphology, enzyme activities, and beneficial bacterial populations. In conclusion, Co-FFM, improves the nutritional value of FM, converting it into a decent source of dietary protein for broilers.

The Chinese medicine Xin-tong-tai granule protects atherosclerosis by regulating oxidative stress through NOX/ROS/NF-κB signal pathway.

BACKGROUND: Xin-tong-tai Granule (XTTG), a traditional Chinese medicine, has been used to treat atherosclerosis (AS), but its mechanism is poorly understood. Intriguingly, oxidative stress has been recognized as vital factors in the treatment of atherosclerosis. PURPOSE: This study aims to explore the potential mechanism of XTTG for treating AS. METHODS: An in-vivo model of AS was established by feeding ApoE-/- mice with a high-fat diet (HFD), and the Human Aortic Vascular Smooth Muscle Cells (HAVSMCs) were induced by oxidized low-density lipoprotein (ox-LDL) in vitro. After treatment, the blood lipid levels and pathological aortic changes of each group were observed, and the cell proliferation and lipid droplet aggregation in each group were evaluated. The oxidative stress indicators such as malondialdehyde (MDA) and superoxide dismutase (SOD) levels and related NOX/ROS/NF-κB signaling pathway indicators were observed. RESULTS: XTTG improved blood lipid levels and pathological aortic changes of ApoE-/- mice with HFD feeding, inhibited HAVSMCs proliferation and lipid droplet aggregation induced by ox-LDL, reduced MDA content, increased SOD content, inhibited NOX4 and p22phox protein expression, downregulated ROS content, inhibited IKK-α, IKK-ß, NF-κB protein and mRNA expression and the phosphorylation of NF-κB. CONCLUSION: XTTG can inhibit NOX/ROS/NF-κB signaling pathway, reduce damages caused by oxidative stress, and exert anti-AS effects.

Anti-oxidant response of lipidom modulates lipid metabolism in Caenorhabditis elegans and in OxLDL-induced human macrophages by tuning inflammatory mediators.

Atherosclerosis is the main pathological process of several cardiovascular diseases. It may begin early in life and stay latent and asymptomatic for an extended period before its clinical manifestation. The formation of foamy macrophages due to dysregulated lipid metabolism is a key event in the development and progression of atherosclerotic plaque. The current pharmacotherapy for atherosclerosis is not able to address multiple aetiologies associated with the disease. Lipidom, an herbal prescription medicine, has anti-oxidant, lipid lowering and anti-inflammatory properties that lead to multifaceted treatment benefits against chronic inflammation, dyslipidaemia, and oxidative stress. The present study aimed to characterize the pharmacological effects of Lipidom using various experimental models. The phytochemical analysis of Lipidom was performed on ultra-high performance liquid chromatography (UHPLC) platform. Lipidom was evaluated for cytosafety, IL-1ß and MCP-1 release, modulation of NLRP3 pathway, NFκB activity, ROS generation, lipid accumulation and gene expression in THP1 macrophages. Furthermore, Lipidom evaluation was also performed in the N2, CF1553, and TJ356 strains of Caenorhabditis elegans (C. elegans). The evaluation of brood size, adult (%), lipid accumulation, triglyceride levels, SOD-3 GFP signal, MDA formation, DAF-16 nuclear translocation, and gene expression was performed in C. elegans. Lipidom treatment significantly reduced the inflammatory mediators, lipid accumulation, oxidative stress, and normalized genes involved in the development of foamy macrophages. Lipidom treated C. elegans showed a significant decline in lipid accumulation and oxidative stress. Taken together, Lipidom treatment showed a multifaceted approach in the modulation of several mediators responsible for the development and progression of atherosclerotic plaque.

The impact of varying doses of moringa leaf methanolic extract supplementation in the cryopreservation media on sperm quality, oxidants, and antioxidant capacity of frozen-thawed ram sperm.

To increase rams' post-thaw semen quality following cryopreservation, this study used enriched Tris-based diluent with varying amounts of moringa leaf methanolic extract (MLME). The antioxidant activity, total phenolic, and total flavonoid content were all assessed in MLME. The sperm of five healthy Awassi rams were collected, divided into 4 equal aliquots, and diluted [1:5; (v/v)] in Tris-citrate-glucose extender supplemented with 0.48, 0.56, and 0.64 mg MLME/ml or without MLME supplementation (control). The percentages of sperm total motility (STM, %), sperm progressive motility (SPM, %) and viability (V, %), abnormal morphology (AM, %), membrane functional integrity (MFI, %), and acrosome integrity (AI %) were measured. Malondialdehyde (MDA), nitric oxide (NO), ascorbic acid (AA), superoxide dismutase (SOD), glutathione peroxidase (GPx), total cholesterol (TC), low-density lipoproteins (LDL), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), zinc (Zn), and copper (Cu) were measured. The total phenolic gallic acid and flavonoid catechin (equivalent) contents were 19.78 mg/g and 11.94 mg/g, respectively. 2,2-Diphenyl-1-picrylhydrazyl (34.37 mM TE/g) and 2,2'-azino-bis/3-ethylbenzothiazoline-6-sulfonic acid (53.47 mM TE/g) were found in MLME. MLME had a 64.59 mM TE/g ferric-reducing power. In comparison to control, the addition of 0.64 mg/ml MLME to Tris-based extender resulted in the highest (P < 0.001) STM (55.22 ± 0.98), SPM (45.41 ± .70), SV (60.01 ± 1.05), MFI (75.23 ± 0.77), and AI (73.13 ± 0.72) and the lowest (P < 0.001) AM (21.34 ± 0.72) values. In comparison to the control, the addition of 0.56 mg/ml semen extender resulted in lower STM, SPM, SV, MFI, and AI with higher AM percentages. MDA (P = 0.03), NO (P = 0.012), CHO (P = 0.0001), and LDL (P = 0.004) were reduced by 0.64 mg/ml MLME, while AA (P = 0.017) and SOD (P = 0.0001) were elevated. In conclusion, the highest copper (P = 0.006) and lowest zinc concentrations in MLME (0.48 mg/ml extender) deteriorated the post-thaw semen quality, prompting us to suggest the addition of 0.64 mg MLME to rams' Tris-based semen extender.

Extracellular vesicle-mediated transfer of lncRNA CLDN10-AS1 aggravates low-density lipoprotein-induced vascular endothelial injury.

Oxidized low-density lipoprotein (ox-LDL) stimulation impairs the oxidation-reduction equilibrium in vascular endothelial cells (VECs) and contributes to atherosclerosis (AS). This study probed the mechanisms of extracellular vesicle (EV)-mediated transfer of lncRNA CLDN10 antisense RNA 1 (CLDN10-AS1) in ox-LDL-induced VEC injury. Initially, VEC injury models were established by treating human umbilical vein endothelial cells (HUVECs) with ox-LDL. EVs were isolated from HUVECs (HUVECs-EVs) and identified. CLDN10-AS1, microRNA (miR)-186, and Yin Yang 1 (YY1) expressions in ox-LDL-treated HUVECs and EVs derived from these cells (ox-EVs) were measured. HUVECs were incubated with EVs, after which the cell viability, apoptosis, and concentrations of proinflammatory cytokines and oxidative stress markers were measured. We discovered that CLDN10-AS1 and YY1 were upregulated in ox-LDL-treated HUVECs, whereas miR-186 was downregulated. ox-EVs treatment elevated CLDN10-AS1 expression in HUVECs and ox-EVs overexpressing CLDN10-AS1 promoted VEC injury. Besides, CLDN10-AS1 is competitively bound to miR-186 and promoted YY1 expression. Rescue experiments revealed that miR-186 overexpression or YY1 suppression partially reversed the roles of ox-EVs overexpressing CLDN10-AS1 in ox-LDL-induced VEC injury. Lastly, clinical serum samples were collected for verification. Overall, CLDN10-AS1 carried by HUVECs-EVs into HUVECs competitively bound to miR-186 to elevate YY1 expression, thereby aggravating ox-LDL-induced VEC injury.

[Study on the mechanism of paeonol in improving low-density lipoprotein-induced human vascular endothelial cell injury].

OBJECTIVE: To investigate the effects of paeonol on low-density lipoprotein-induced human vascular endothelial cell injury and its molecular mechanisms. METHODS: Human umbilical vein endothelial cells (HUVECs) were divided into 9 groups, normal control (NC) group, ox-LDL group (100 ng/L ox-LDL), low, medium, and high-dose paeonol groups (60 µmol/L, 120 µmol/L, 240 µmol/L paeonol+100 ng/L ox-LDL), ox-LDL+small interfering RNA negative control (si-NC) group, ox-LDL+circ_0003204 small interfering RNA (si-circ_0003204) group, middle dose group+ox-LDL+circ_0003204 overexpression negative control (pcDNA-NC) group, middle dose group+ox-LDL+circ_0003204 overexpression (pcDNA-circ_0003204) group, three replicate wells in each group. MTT flow cytometry, and Western blot were used to detect cell proliferation, apoptosis and protein (CDK2, Bcl2, p27, Bax) expressions, respectively. Malondialdehyde (MDA) and superoxide dismutase (SOD) kit were used to detect MDA content and SOD activity; real-time quantitative PCR (RT-qPCR) was used to detect the expression of circ_0003204. RESULTS: Compared with the NC group, the proliferation activity, protein expressions of CDK2 and Bcl2, and SOD activity of HUVECs in the ox-LDL group were decreased significantly (P＜0.05), and the apoptosis rate, protein expressions of p27 and Bax, MDA content, and circ_0003204 expression were increased significantly (P＜ 0.05). Compared with the ox-LDL group, the proliferation activity, protein (CDK2, Bcl2) expressions and SOD activity of HUVECs in the low, medium and high dose paeonol groups were increased significantly (P＜0.05), and the apoptosis rate, protein (p27, Bax) expressions, MDA content And circ_0003204 expression were decreased significantly (P＜ 0.05). Compared with ox-LDL+si-NC group, the proliferation activity, protein (CDK2, Bcl2) expressions, SOD activity of HUVECs in ox-LDL+si-circ_0003204 group were increased significantly (P＜0.05), the apoptosis rate, protein (p27, Bax) expressions, and the content of MDA were decreased significantly (P＜0.05). Compared with the middle-dose+ox-LDL+pcDNA-NC group, the HUVECs proliferation activity, protein (CDK2, Bcl2) expressions, and SOD activity in the middle-dose+ox-LDL+pcDNA-circ_0003204 group were decreased significantly (P＜0.05), and the levels of circ_0003204, apoptosis rate, protein (p27, Bax) expressions and MDA content were increased significantly (P＜0.05). CONCLUSION: Paeonol can inhibit ox-LDL-induced apoptosis and oxidative stress of human umbilical vein endothelial cells, and alleviate human umbilical vein endothelial cell injury. The mechanism of action may be related to the down-regulation of circ_0003204 expression.

Oral administration of potato peel extract affects serum blood metabolites, liver function and ameliorating oxidative stress induced in rabbits exposed to cold stress.

This study investigated the effect of potato peel extract (PPE), orally administrated to rabbits, on serum blood metabolites and ameliorating oxidative stress induced by cold stress under Egyptian winter conditions. Twenty-four bucks grouped into three treatments (8 animals per group) were used for the experiment. The animals received 1.5 ml of water orally, containing 0 (PPE0), 25 (PPE25) or 50 (PPE50) mg PPE/kg live weight. Bucks were randomly assigned into three homogenous equal groups according to the level of PPE. Treatments were applied to each animal every two days over a period of three months including one month as an adaptation period. At the 8th week of the experiment, blood samples were collected from each buck and at the end of the experiment, bucks were slaughtered, and some organs were collected and weighed. The PPE improved (p < 0.05) blood total protein, albumin, globulin and glucose. The blood concentration of total lipid, cholesterol, triglyceride, low density lipoprotein and very low-density lipoprotein (were increased (p < 0.02) in PPE rabbits. Furthermore, PPE extract doses decreased (p < 0.001) oxidant thiobarbituric reactive substance (TBARS) in both blood and liver. Other liver and blood antioxidant system enzymes such as catalase, glutathione peroxidase, and superoxide dismutase were improved (p < 0.005) by PPE supplementation. Overall, oral administration of PPE up to 50 mg/kg live weight can have positive effects on rabbit health under cold stress.

Protective Effects of Medicinal Plant Decoctions on Macrophages in the Context of Atherosclerosis.

Atherosclerosis is a hallmark of most cardiovascular diseases. The implication of macrophages in this pathology is widely documented, notably for their contribution to lipid accumulation within the arterial wall, associated with oxidative stress and inflammation processes. In order to prevent or limit the atherosclerosis damage, nutritional approaches and medicinal plant-based therapies need to be considered. In Reunion Island, medicinal plant-based beverages are traditionally used for their antioxidant, lipid-lowering and anti-inflammatory properties. The aim of our study was to assess the protective effects of eight medicinal plant decoctions in an in vitro model of RAW 264.7 murine macrophages exposed to pro-atherogenic conditions (oxidized low-density lipoproteins-ox-LDL-E. coli Lipopolysaccharides-LPS). The impact of polyphenol-rich medicinal plant decoctions on cell viability was evaluated by Neutral Red assay. Fluorescent ox-LDL uptake was assessed by flow cytometry and confocal microscopy. Activation of NF-κB was evaluated by quantification of secreted alkaline phosphatase in RAW-Blue™ macrophages. Our results show that medicinal plant decoctions limited the cytotoxicity induced by ox-LDL on macrophages. Flow cytometry analysis in macrophages demonstrated that medicinal plant decoctions from S. cumini and P. mauritianum decreased ox-LDL uptake and accumulation by more than 70%. In addition, medicinal plant decoctions also inhibited NF-κB pathway activation in the presence of pro-inflammatory concentrations of E. coli LPS. Our data suggest that medicinal plant decoctions exert protective effects on ox-LDL-induced cytotoxicity and limited macrophage lipid uptake. Moreover, herbal preparations displayed anti-inflammatory properties on macrophages that can be of interest for limiting the atherosclerotic process.

Protective activities of distinct omega-3 enriched oils are linked to their ability to upregulate specialized pro-resolving mediators.

Clinical studies using a range of omega-3 supplements have yielded conflicting results on their efficacy to control inflammation. Omega-3 fatty acids are substrate for the formation of potent immune-protective mediators, termed as specialized pro-resolving mediators (SPM). Herein, we investigated whether observed differences in the potencies of distinct omega-3 supplements were linked with their ability to upregulate SPM formation. Using lipid mediator profiling we found that four commercially available supplements conferred a unique SPM signature profile to human macrophages, with the overall increases in SPM concentrations being different between the four supplements. These increases in SPM concentrations were linked with an upregulation of macrophage phagocytosis and a decreased uptake of oxidized low-density lipoproteins. Pharmacological inhibition of two key SPM biosynthetic enzymes 5-Lipoxygenase or 15-Lipoxygenase reversed the macrophage-directed actions of each of the omega-3 supplements. Furthermore, administration of the two supplements that most potently upregulated macrophage SPM formation and reprogrammed their responses in vitro, to APOE-/- mice fed a western diet, increased plasma SPM concentrations and reduced vascular inflammation. Together these findings support the utility of SPM as potential prognostic markers in determining the utility of a given supplement to regulate macrophage responses and inflammation.

Shen-Hong-Tong-Luo Formula Attenuates Macrophage Inflammation and Lipid Accumulation through the Activation of the PPAR-γ/LXR-α/ABCA1 Pathway.

Atherosclerosis (AS) is the killer of human health and longevity, which is majorly caused by oxidized lipoproteins that attack macrophages in the endarterium. The Shen-Hong-Tong-Luo (SHTL) formula has shown great clinical efficacy and vascular protective effect for over 30 years in China, to attenuate AS progression. However, its pharmacological mechanism needs more investigation. In this study, we first investigated the chemical composition of SHTL by fingerprint analysis using high-performance liquid chromatography. In primary mouse peritoneal macrophages induced by lipopolysaccharide (LPS), we found that SHTL pretreatment suppressed reactive oxygen species accumulation and reversed the increases of the inflammatory factors, TNF-α and IL-6. Moreover, lipid accumulation induced by oxidized low-density lipoprotein (Ox-LDL) in macrophages was inhibited by SHTL. Additionally, network pharmacology was used to predict the potential targets of SHTL as the PPAR-γ/LXR-α/ABCA1 signaling pathway, which was validated in macrophages and ApoE-/- mice by histopathological staining, qPCR, and Western blot analysis. Importantly, the protective effect of SHTL in the LPS- and Ox-LDL-induced macrophages against inflammation and lipid accumulation was attenuated by GW9662, a PPAR-γ antagonist, which confirmed the prediction results of network pharmacology. In summary, these results indicated that SHTL pretreatment reduced inflammation and lipid accumulation of macrophages by activating the PPAR-γ/LXR-α/ABCA1 pathway, which may provide a new insight into the mechanism of SHTL in the suppression of AS progression.

Vitamin D inhibits Tissue Factor and CAMs expression in oxidized low-density lipoproteins-treated human endothelial cells by modulating NF-κB pathway.

Epidemiologic studies have clearly demonstrated the correlation existing between Vitamin D (Vit. D) deficiency and increased risk of developing cardiovascular disease, suggesting that it might have a protective role in this clinical setting. Although many experimental studies have investigated the molecular mechanisms by which Vit. D might exert these effects, its potential role in protecting against athero-thrombosis is still partially unknown. We have investigated whether Vit. D might exert anti athero-thombotic effects by preventing expression of adhesion molecules (CAMs) and Tissue Factor (TF), molecules involved in atherothrombotic pathophysiology, in oxLDL stimulated endothelial cells (HUVEC). Moreover, we have investigated whether Vit. D effects might be due to the NF-kB modulation. HUVEC cultivated in medium enriched with Vit. D (10 nM) were stimulated with oxLDL (50 µg/ml). TF gene (RT-PCR), protein (Western blot), surface expression (FACS) and procoagulant activity (FXa generation assay) were measured. Similarly, CAMs gene (RT-PCR), surface expression (FACS) and soluble values (ELISA) were measured. NF-kB translocation was also investigated. Vit. D significantly reduced TF gene as well protein expression and procoagulant activity in oxLDL-treated HUVEC. Similar effects were observed for CAMs. These effects were associated with Vit. D modulation of NF-κB pathway. This study, although in vitro, indicate that Vit. D has protective effect on endothelial cells by inhibiting expression of TF and CAMs, proteins involved in atherothrombotic pathophysiology. Further studies will be necessary to translate these findings to a clinical scenario to better define the potential therapeutical role of Vit. D supplementation in the management of cardiovascular disease in patients with Vit. D deficiency.

Bushen Kangshuai tablet inhibits progression of atherosclerosis by intervening in macrophage autophagy and polarization.

OBJECTIVE: To investigate the efficacy of Bushen Kangshuai (BS-KS) tablet on autophagy and polarization in mouse macrophage RAW 264.7. MEYHODS: Macrophage autophagy was induced by oxidized low-density lipoprotein (100 µg/mL). To detect the levels of autophagy, macrophage were transfected with double fluorescence LC3 autophagy adenovirus, then the numbers of autophagosomes and autophagic lysosomes were asessed by confocal microscopy. The autophagy related proteins expression of PI3K, Akt, phospho-mAkt (p-Akt) and mTOR, phospho-mTOR ([p-TOR), p62, microtubule-associated protein 1 (LC3-Ⅱ)were determined by western blotting. The macrophage polarization model was induced by lipopolysaccharide (1 µg/mL). The mRNA levels of iNOS, CD86 (M1 macrophages marker molecules), and CD206, Arg-1 (M2 macrophages marker molecules) were detected by real-time quantitative PCR. The concentration of cytokines TNF-α and IL-10 was determined by enzyme-linked immunosorbent assay. The protein expression of nuclear proteins PPAR-γ, NF-κB, and cytoplasmic protein IKB α was determined by western blotting. RESULTS: The expression of the autophagy-related protein LC3-Ⅱ was increased and the expression of p62 was decreased in the BS-KS intervention group. The protein expression of PI3K, p-Akt, and p-mTOR was also reduced. BS-KS also inhibited the mRNA expression of iNOS and CD86 on M2 macrophage, but promoted the expression of CD206 and Arg-1 on M2 macrophage. With respect to the regulation of inflammatory factors, BS-KS could inhibit the secretion of pro-inflammatory TNF-α and promote the secretion of anti-inflammatory IL-10. It also inhibited the protein expression of IKB-α and NF-κB, and promoted the expression of nuclear protein PPAR-γ. CONCLUSION: We believe that BS-KS promotes macrophage autophagy by increasing the level of autophagy protein and inhibiting the PI3K/Akt/mTOR signaling pathway. Furthermore, BS-KS seems to inhibit macrophage M1 polarization and promote M2 polarization via the PPAR gamma /NF-κB signaling pathway, thus playing an inhibitory role in atherosclerosis.

L-Arginine Ameliorates High-Fat Diet-Induced Atherosclerosis by Downregulating miR-221.

OBJECTIVES: Atherosclerosis (AS) is a severe disease in which the inside of an artery narrows because of plaque formation, leading to endothelial injury in the patients. Although it has been found that endothelial nitric oxide synthase (eNOS), which produces a low concentration of NO, is necessary for endothelial function and integrity, the regulatory mechanisms of eNOS expression against the pathogenesis and development of AS are unclear. Evidence has indicated that diet supplementation with L-arginine could reduce the size of the endothelial injury lesions in AS patients. In addition, nonencoding microRNAs (miRNAs) were found to be a promising tool that regulates the expression of eNOS in human endothelial cells. DESIGN: The aim of this research was to explore the role of L-arginine in the development of AS and the mechanisms by which miR-221 influences the possible signaling pathways in endothelial cells during AS. RESULTS: The results suggested that L-arginine could prevent oxidized low-density lipoprotein-induced apoptosis in endothelial cells, which is associated with the downregulation of miR-221. Similar results were also observed in rat AS models. CONCLUSION: This research could provide potential therapies for the treatment of AS.

Expression of chemokines in macrophage polarization and downregulation of NFκB in aorta allow macrophage polarization by diosgenin in atherosclerosis.

M1 macrophages serve one edge as proinflammatory and M2 macrophages serve the other edge as an anti-inflammatory macrophage. It appears that a related "switch" in macrophage morphology may also happen in the course of atherosclerosis, which has not yet been elucidated. An atherogenic diet (AD) was given to rats, and induction of macrophage differentiation and the nuclear localization of nuclear factor-kappa B (NFκB) were investigated by Western blot and immunofluorescence. Chemokines were analyzed using an antibody array with 32 target proteins. M2 macrophage transformation was confirmed in diosgenin-treated aorta by immunofluorescence and was validated in vitro using THP-1 cells. MAC387 (macrophage marker) and NFκBp65 (inflammatory hub) were upregulated in oxidatively-modified low-density lipoprotein (OxyLDL) and AD-induced condition. Macrophage differentiation, which induced the formation of inflammatory mediators, was not significantly suppressed by the inhibition of NFκB using dexamethasone. M1 macrophage polarization was identified in OxyLDL-induced monocytes, which are proinflammatory in nature, whereas M2 macrophage polarization was noticed in diosgenin-treated monocytes, which exhibit anti-inflammatory properties. M1-and M2-specific chemokines were analyzed using chemokine antibody array. Furthermore, the expression of proinflammatory macrophage (M1) was noticed in AD-induced aorta and anti-inflammatory macrophage (M2) was observed in diosgenin-treated aorta. This is the first report where, unifying the mechanism of diosgenin as aan nti-atherosclerotic and the expression of M1 and M2 specific chemokines is shown by downregulating NFκB and not by preventing the differentiation of monocyte into a macrophage, but by allowing macrophage to differentiate into M2, which aids in preventing the atherosclerotic progression.

Terminalia Arjuna bark extract impedes foam cell formation and promotes apoptosis in ox-LDL-stimulated macrophages by enhancing UPR-CHOP pathway.

BACKGROUND: Increased macrophage and foam cell apoptosis during early atherogenesis retards plaque progression by impeding foam cell formation, suppressing inflammation and limiting lesion cellularity. Our previous in vitro study in THP1 macrophages demonstrated that Terminalia Arjuna (TA) attenuates dual-specificity phosphatase1 (DUSP1), a key negative regulator of JNK/P38MAPK signaling cascade, the branch also implicated in the UPR (unfolded protein response)-CHOP-mediated apoptotic pathway; however this pathway has not been explored so far in the presence of TA. Therefore, we aimed to elucidate the pro-apoptotic effect of aqueous bark extract of TA (aqTAE) on macrophage and foam cells and the underlying mechanism associated with it. METHODS: THP1 cells were initially differentiated into macrophages with phorbol-12-myristate-13-acetate (PMA) (100 ng/ml) for 24 h, followed by ox-LDL (100 µg/ml) treatment for another 24 h to induce foam cell formation. Thereafter, macrophages and ox-LDL- treated cells were incubated with aqTAE (100 µg/ml) for the next 24 h. Further, Oil Red O (ORO) staining, CD36 expression profiling, apoptotic assay and transcriptional and translational expression of ER-stress markers i.e., X-box binding protein 1 (XBP1) and C/EBP homologous protein (CHOP) were performed for elucidating the potential mechanism underlying TA-induced macrophage and foam cell apoptosis. RESULTS: We demonstrated that ox-LDL treatment significantly increased lipid accumulation and upregulated CD36 expression, indicating foam cell formation; while the addition of aqTAE resulted in a significant decline in ORO positive cells, and suppression of CD36 expression in ox-LDL-stimulated macrophages, suggestive of reduced formation of lipid-laden foam cells. Further, aqTAE treatment alone and in combination with oxidized low-density lipoprotein (ox-LDL) stimulus, significantly attenuated CD36 expression; increased apoptosis; and augmented the expression of UPR regulatory proteins including XBP1 and CHOP, and similar observations were noted when cells were treated with ox-LDL alone. These findings indicate that TA promotes macrophage and foam cell apoptosis via enhancing UPR-mediated activation of JNK/p38MAPK-CHOP pathway in a DUSP1-dependent manner, implying a possible interplay between ox-LDL-induced ER stress- and TA-mediated MAPK signaling. CONCLUSION: Our data shows that aqTAE inhibits foam cell formation, as well as promotes macrophage and foam cell apoptosis by augmenting UPR- JNK/p38MAPK-CHOP signaling cascade via inhibiting DUSP1. These findings provide novel mechanistic insight into the anti-atherogenic potential of TA, which may prove beneficial against early-stage atherosclerotic lesions.

Effects of oral butyrate supplementation on inflammatory potential of circulating peripheral blood mononuclear cells in healthy and obese males.

Sodium butyrate is well-known for its immune-modulatory properties. Studies until now only focused on the in vitro effects of butyrate or assessed local effects in the gut upon butyrate administration. In this trial, we studied the systemic anti-inflammatory effects induced by sodium butyrate supplementation in humans. Nine healthy (Lean) and ten obese (metabolic syndrome group, MetSyn) males were given 4 grams sodium butyrate daily for 4 weeks. PBMCs were isolated before and after supplementation for direct stimulation experiments and induction of trained immunity by oxidized low-density lipoprotein (oxLDL), ß-glucan, or Bacillus Calmette-Guérin vaccine (BCG). Butyrate supplementation moderately affected some of the cytokine responses in the MetSyn group. In the direct stimulation setup, effects of butyrate supplementation were limited. Interestingly, butyrate supplementation decreased oxLDL-induced trained immunity in the MetSyn group for LPS-induced IL-6 responses and Pam3CSK4-induced TNF-α responses. Induction of trained immunity by ß-glucan was decreased by butyrate in the MetSyn group for Pam3CSK4-induced IL-10 production. In this study, while having only limited effects on the direct stimulation of cytokine production, butyrate supplementation significantly affected trained immunity in monocytes of obese individuals with metabolic complications. Therefore, oral butyrate supplementation may be beneficial in reducing the overall inflammatory status of circulating monocytes in patients with metabolic syndrome.

Baicalin inhibits proliferation and promotes apoptosis of vascular smooth muscle cells by regulating the MEG3/p53 pathway following treatment with ox­LDL.

Atherosclerosis (AS) is a systemic disease associated with lipid metabolic disorders and abnormal proliferation of smooth muscle cells. Baicalin is a flavonoid compound isolated from the dry roots of Scutellaria baicalensis Georgi and exerts anti­proliferative effects in various types of cells. However, the effect of baicalin on AS remains unclear. In the present study, serum samples were collected from patients with AS and an in vitro model of AS was established using oxidized low­density lipoprotein (ox­LDL)­treated human aorta vascular smooth muscle cells (HA­VSMCs). The siRNA transfection and overexpression efficiency of endogenous maternally expressed gene 3 (MEG3) and the expression level of MEG3 were analyzed by reverse transcription­quantitative polymerase chain reaction (RT­qPCR). The effects of alterations in expression levels of MEG3 were assessed by MTT assay, bromodeoxyuridine incorporation assay, 5­ethynyl­2'­deoxyuridine staining, wound healing assay, immunofluorescence and western blotting in HA­VSMCs. qPCR indicated that the expression of MEG3 was reduced in serum samples from patients with AS and ox­LDL­treated HA­VSMCs, compared with serum samples from healthy patients and untreated HA­VSMCs, respectively. Further experiments indicated that ox­LDL­induced decrease of MEG3 expression was reversed by treatment with baicalin in a concentration­dependent manner. Following treatment with ox­LDL, decreased expression of MEG3 promoted proliferation and migration, and suppressed apoptosis in HA­VSMCs. Furthermore, treatment with baicalin reversed these effects on proliferation and apoptosis in ox­LDL­treated HA­VSMCs. The current study indicated that downregulated expression of MEG3 increased cell cycle­associated protein expression. However, treatment with baicalin inhibited the expression of cell­cycle associated proteins in HA­VSMCs with MEG3 knockdown. In addition, baicalin activated the p53 signaling pathway and promoted the expression and transport of p53 from the cytoplasm to nucleus following MEG3 knockdown in ox­LDL­treated HA­VSMCs. Baicalin inhibited proliferation and promoted apoptosis by regulating the expression of MEG3/p53, indicating that baicalin may serve a role in AS by activating the MEG3/p53 signaling pathway. The present study suggested a potential mechanism underlying the protective role of baicalin in the in vitro model of AS, and these results may be used to develop novel therapeutic approaches for the affected patients.

Primary prevention of atherosclerosis by pretreatment of low-density lipoprotein receptor knockout mice with sesame oil and its aqueous components.

Pharmacological intervention using statins and PCSK9 inhibitors have become first-line therapy in the prevention of hypercholesterolemia and atherosclerosis. Currently, no agent is available for the primary prevention of atherosclerosis. However, there is an emerging hypothesis that atherosclerosis could be driven by inflammation. In this study, we tested whether pretreatment with an aqueous extract from sesame oil (SOAE), which showed potent anti-inflammatory properties without hypocholesterolemic actions, would prevent subsequent atherosclerosis development in a mouse model. RAW 264.7 macrophages and female low-density lipoprotein receptor knockout (LDLR-/-) mice were used for in vitro and in vivo studies, respectively. Plasma lipids, cytokines and atherosclerotic lesions were quantified at the end of the study. RNA was extracted from the liver and aortic tissues and used for gene analysis. Pre-treatment of SOAE prevented Ox-LDL uptake by RAW macrophages and further inflammation in vitro. SOAE pre-treatment significantly reduced atherosclerotic lesions and pro-inflammatory gene expressions in LDLR-/- mice as compared to control mice. No significant change in plasma cholesterol levels was observed. A significant reduction in plasma levels of TNF-α, IL-6, MCP-1 and VCAM1 was observed in the SOAE pre-treated animals. This is the first study that demonstrates that pre-treatment with anti-inflammatory agents, could delay/decrease atherosclerosis.

Selected Phytoestrogens Distinguish Roles of ERα Transactivation and Ligand Binding for Anti-Inflammatory Activity.

Estrogen receptor α (ERα) is a ligand-activated transcriptional activator that is also involved vascular inflammation and atherosclerosis. Whether different ligands may affect this activity has not been explored. We screened a panel of phytoestrogens for their role in ERα binding and transcriptional transcription, and correlated the findings to anti-inflammatory activities in vascular endothelial cells stably expressing either a wild-type or mutant form of ERα deficient in its membrane association. Taxifolin and silymarin were "high binders" for ERα ligand binding; quercetin and curcumin were "high activators" for ERα transactivation. Using these phytoestrogens as functional probes, we found, in endothelial cells expressing wild-type ERα, the ERα high activator, but not the ERα high binder, promoted ERα nuclear translocation, estrogen response element (ERE) reporter activity, and the downstream gene expression. In endothelial cells expressing membrane association-deficient mutant ERα, the ERα nuclear translocation was significantly enhanced by taxifolin and silymarin, which still failed to activate ERα. Inflammation response was examined using the systemic or vascular inflammation inducers lipopolysaccharide or oxidized low-density lipoprotein. In both cases, only the ERα high activator inhibited nuclear translocation of nuclear factor κB, JNK, and p38, and the production of inflammatory cytokines IL-1ß and TNFα. We confirm a threshold nuclear accumulation of ERα is necessary for its transactivation. The anti-inflammatory activity of phytoestrogens is highly dependent on ERα transactivation, less so on the ligand binding, and independent of its membrane association. A pre-examination of phytoestrogens for their mode of ERα interaction could facilitate their development as better targeted receptor modifiers.

Alantolactone suppresses APOC3 expression and alters lipid homeostasis in L02 liver cells.

A high level of APOC3 expression is an independent risk factor for some lipid metabolism-related diseases, such as cardiovascular disease (CVD), nonalcoholic fatty liver disease (NAFLD) and atherosclerosis (AS). This suggests that down-regulating APOC3 expression is a potential way of regulating lipid levels. In this study, we used luciferase reporter screening to identify a natural compound, alantolactone (ALA), that can inhibit the promoter activity of APOC3. ALA decreased APOC3 expression at both mRNA and protein levels. Then we pretreated L02 liver cells with oxLDL to investigate the function of ALA in lipid homeostasis. Intriguingly, ALA attenuated oxLDL-induced foam cell formation by reducing total cholesterol (TC) and triglyceride (TG) contents. Furthermore, these results could be reversed by overexpressing APOC3 protein. ALA inhibited tyrosine phosphorylation (Tyr705pho) of STAT3 to down-regulate APOC3 expression. Intriguingly, overexpression of a wild-type STAT3 or a constitutively active form of STAT3 (STAT3-C) up-regulated APOC3 expression and partly reversed the effect of ALA in oxLDL-induced L02 cells. Overexpression of wild-type STAT3 also increased TC but not TG contents in L02 cells. However, overexpression of STAT3-C significantly increased TC and TG contents, and the effect of ALA was partly attenuated by STAT3-C, although this was not statistically significant. These results suggest that ALA attenuates lipid accumulation through down-regulation of APOC3 expression, at least in part by inhibiting STAT3 signaling.