Development and validation of a method to deliver vitamin A to macrophages.

Macrophages are critical players in the development of atherosclerotic lesions, where they promote local and systemic inflammation. Macrophages engulf lipoproteins and cell debris upon entry into the arterial wall, becoming lipid-laden foam cells. While most lipids found in foam cells are triglyceride and cholesterol, these cells accumulate several other lipids with bioactive properties, such as vitamin A and carotenoids. Vitamin A has strong immunomodulatory actions in macrophages and other immune cells. For example, macrophages release vitamin A as retinoic acid to modulate T cell differentiation, but the implication of intracellular vitamin A stores in this process remains elusive due to the lack of an adequate experimental model to load vitamin A into macrophages. The purpose of this study was to develop a reliable method to deliver vitamin A to cultured murine macrophages. Our results show that thioglycolate-elicited peritoneal macrophages fail to take up significant levels of vitamin A when provided as free retinol. Cultured macrophages and macrophages in the peritoneal cavity can take up retinyl esters, either as retinyl ester-loaded serum or retinyl esters infused directly into the peritoneal cavity. HPLC analyses in macrophage lysates revealed that the intraperitoneal injection method results in a fourfold greater vitamin A loading efficiency than retinyl ester-loaded serum added to cultured cells. These two alternative methods provide an efficient and reliable methodology to load macrophages with vitamin A for downstream applications such as studies of gene regulation trafficking of intracellular vitamin A, and vitamin A release from macrophages.

Mechanical Stress Modulates Calcium-Activated-Chloride Currents in Differentiating Lens Cells.

During accommodation, the lens changes focus by altering its shape following contraction and relaxation of the ciliary muscle. At the cellular level, these changes in shape may be accompanied by fluid flow in and out of individual lens cells. We tested the hypothesis that some of this flow might be directly modulated by pressure-activated channels. In particular, we used the whole cell patch clamp technique to test whether calcium-activated-chloride channels (CaCCs) expressed in differentiating lens cells are activated by mechanical stimulation. Our results show that mechanical stress, produced by focally perfusing the lens cell at a constant rate, caused a significant increase in a chloride current that could be fully reversed by stopping perfusion. The time course of activation and recovery from activation of the flow-induced current occurred rapidly over a time frame similar to that of accommodation. The flow-induced current could be inhibited by the TMEM16A specific CaCC blocker, Ani9, suggesting that the affected current was predominantly due to TMEM16A chloride channels. The mechanism of action of mechanical stress did not appear to involve calcium influx through other mechanosensitive ion channels since removal of calcium from the bath solution failed to block the flow-induced chloride current. In conclusion, our results suggest that CaCCs in the lens can be rapidly and reversibly modulated by mechanical stress, consistent with their participation in regulation of volume in this organ.

Dietary n-3 but not n-6 fatty acids modulate anthropometry and fertility indices in high-fat diet fed rats: a two-generation study.

The present study assessed the modulatory potentials of dietary n-3 [α-linolenic acid (ALA, 18:3n-3, eicosapentaenoic acid (EPA, 20:5n-3 + docosahexaenoic acid (DHA) 22:6n-3), and n-6 fatty acid (LA, 18:2n-6)] on anthropometric parameters and fertility indices in high-fat-fed rats. Weanling female Wistar rats were fed with control diet (7% lard), high-fat diet (35% lard, HFL), high-fat with fish oil (21% fish oil + 14% lard, HFF), high-fat with canola oil (21% canola oil + 14% lard, HFC) and high-fat with sunflower oil (21% sunflower oil + 14% lard, HFS) for 2 months, mated and continued on their diets during pregnancy. At gestation day 18-20, the intra-uterine environment was examined in representative rats, and the rest were allowed for delivering pups. The pups after lactation were subjected to mating and feeding trials as above. Growth parameters (body weight, body length (BL), abdominal circumference (AC), thoracic circumference (TC), and Lee index and fertility parameters (litter size and sex ratio) were studied. Feeding HFL diet increased BL (16%), AC (33%) and TC (21%) compared to control (p < 0.05). Adipose tissue accumulation was 11% higher in the HFL group compared to control and was lowered with n-3 fatty acid incorporation in the diet. HFL group exhibited a lower percentage of fertility, pregnancy, and delivery indices. Litter size was decreased by 20%, and litter weight was increased by 23% in HFL group compared to control with more male pups. Our study indicated that n-3 to a larger extent than n-6 fatty acids modulated high-fat induced changes in the anthropometric parameters and fertility indices.

Role of n-3 Fatty Acids on Bile Acid Metabolism and Transport in Dyslipidemia: A Review.

Dietary n-3 fatty acids, especially of marine origin, eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3), have always been lauded for their profound effects on regulating the risk factors for major metabolic disorders. Yet, their consumption rate is poor compared to n-6 fatty acids [linoleic acid (18:2n-6)], which are predominantly consumed. Hence, the skewed n-6 to n-3 fatty acid ratio may have a bearing on the risk factors of various diseases, including dyslipidemia. Dyslipidemia and other lifestyle diseases associated with it, such as diabetes, obesity, hypertension, are a growing concern in both developed and developing countries. A common strategy for addressing dyslipidemia involves bile acid (BA) sequestration, to interrupt the enterohepatic circulation of BA, resulting in the modulation of lipid absorption in the intestine, thereby normalizing the levels of circulating lipids. The BA homeostasis is under the tight control of hepatic and enteric BA transporters. Many investigations have reported the effects of dietary constituents, including certain fatty acids on the reabsorption and transport of BA. However, a critical review of the effects of n-3 fatty acids on BA metabolism and transport is not available. The present review attempts to explore certain unmapped facets of the n-3 fatty acids on BA metabolism and transport in dyslipidemia, and their interplay with biological processes involving lipid rafts and gut microbiome.

Ginger and turmeric lipid-solubles attenuate heated oil-induced oxidative stress in the brain via the upregulation of NRF2 and improve cognitive function in rats.

In this study, we elucidated the modulatory potentials of lipid-solubles from ginger and turmeric that may migrate to oils during heating on the brain antioxidant defense and cognitive response in rats. Male Wistar rats were fed with control diet [including native canola oil (N-CNO), and native sunflower oil (N-SFO)], or experimental diets [including heated canola oil (H-CNO), heated sunflower oil (H-SFO), heated canola oil with ginger (H-CNO + GI), heated canola oil with turmeric (H-CNO + TU), heated sunflower oil with ginger (H-SFO + GI), heated sunflower oil with turmeric (H-SFO + TU)] for 90 days. Memory parameters [Morris water maze, elevated plus maze, novel object recognition test, T-maze (spontaneous alteration)], locomotor skills (open field test and rotarod test), antioxidant defense enzymes, reactive oxygen species, NOS2, ICAM-1, and NRF-2 level in the brain were assessed. Compared to their respective controls, heated oil-fed rats, but not those fed oils heated with ginger or turmeric, showed significant (p < 0.05) reduction in the memory, motor coordination skills, antioxidant defense enzymes, and NRF-2 activation in the brain. Compared to their respective controls, the brain NOS-2 and ICAM-1 were significantly (p < 0.05) increased in heated oil-fed rats, but not those fed oils heated with ginger or turmeric. Chronic intake of repeatedly heated oil causes brain dysfunction by inducing oxidative stress through NRF-2 downregulation. Lipid-solubles from ginger and turmeric that may migrate to oil during heating prevent the oxidative stress and cognitive dysfunction triggered by heated oils in rats.

Ginger and turmeric lipid-solubles attenuate heated oil-induced hepatic inflammation via the downregulation of NF-kB in rats.

PURPOSE: Reusing deep-fried vegetable oils multiple times is a common practice to save costs, and their chronic consumption may cause hepatic dysfunction. In this investigation, we assessed the modulatory effects of ginger and turmeric lipid-solubles that may migrate to oils during heating on the hepatic inflammatory response in rats. METHODS: Male Wistar rats were fed with; 1) control {native canola (N-CNO) or native sunflower (N-SFO)} oil, 2) heated (heated canola {(H-CNO) or heated sunflower (H-SFO)} oil, and 3) heated oil with ginger or turmeric {heated canola with ginger (H-CNO + GI) or heated canola oil with turmeric (H-CNO + TU), heated sunflower oil with ginger (H-SFO + GI) or heated sunflower oil with turmeric (H-SFO + TU)} for 120 days. Hepatic inflammatory response comprising eicosanoids, cytokines, and NF-kB were assessed. RESULTS: Compared to respective controls, feeding heated oils significantly (p < 0.05); 1) increased eicosanoids (PGE2, LTB4, and LTC4) and cytokines (TNF-α, MCP-1, IL-1ß, and IL-6), 2) increased nuclear translocation of NF-kB in the liver, and 3) increased the hepatic expression of 5-LOX, COX-2, BLT-1, and EP-4. However, feeding oils heated with ginger or turmeric positively countered the changes induced by consumption of heated oils. CONCLUSIONS: Consumption of repeatedly heated oil may cause hepatic dysfunction by inducing inflammatory stress through NF-kB upregulation. Lipid-solubles from ginger and turmeric that may migrate to oil during heating prevent the hepatic inflammatory response triggered by heated oils in rats.

Ginger and turmeric lipid-solubles attenuate heated oil-induced cardio-hepatic oxidative stress via the up-regulation of nuclear factor erythroid 2-related factor 2 and decrease blood pressure in rats.

Deep-fried vegetable oils are reused multiple times to save costs, and their chronic consumption may cause organ dysfunction. In this study, we assessed the modulatory effects of lipid-solubles from ginger and turmeric that may migrate to oils during heating, on the cardio-hepatic antioxidant defence response and blood pressure in rats. Male Wistar rats were fed with: (1) control (native rapeseed (N-CNO) or native sunflower (N-SFO)) oil, (2) heated (heated rapeseed (H-CNO) or heated sunflower (H-SFO)) oil and (3) heated oil with ginger or turmeric (heated rapeseed oil with ginger (H-CNO + GI) or heated rapeseed oil with turmeric (H-CNO + TU), heated sunflower oil with ginger (H-SFO + GI) or heated sunflower oil with turmeric (H-SFO + TU)) for 120 d. Oxidative stress (OS) markers, antioxidant enzymes, nitric oxide synthase-2 (NOS-2), intercellular adhesion molecule-1 (ICAM-1), nuclear factor erythroid 2-related factor 2 (NRF-2), markers of hepatic and cardiac function and blood pressure were assessed. Feeding heated oils (H-CNO or H-SFO) (1) increased OS markers, NOS-2 and ICAM-1 expression; (2) decreased antioxidant enzyme activity and NRF-2 level; (3) increased marker enzymes of hepatic and cardiac function and (4) increased systolic and diastolic blood pressure significantly (P < 0·05), when compared with respective native oils (N-CNO or N-SFO). However, feeding oils heated with ginger or turmeric positively countered the changes induced by heated oils. Consumption of repeatedly heated oil causes cardio-hepatic dysfunction by inducing OS through NRF-2 down-regulation. Lipid-solubles from ginger and turmeric that may migrate to oil during heating prevent the oxidative stress and blood pressure triggered by heated oils in rats.

Hyperlipidemia Downregulate Brain Antioxidant Defense Enzymes and Neurotrophins in Rats: Assessment of the Modulatory Potential of EPA+DHA and Zerumbone.

BACKGROUND: Oxidative stress (OS) plays a vital role in the pathogenesis of cognitive disorders. In this study, brain antioxidant defense dysregulation as a consequence of hyperlipidemia, and the efficacy of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA), and zerumbone (Z) in their modulation are assessed. METHODS AND RESULTS: Male Wistar rats are fed control, high-fat (HF), HF + fish oil (HF+F), HF + zerumbone (HF+Z), and HF + fish oil + zerumbone (HF+F+Z) diet for 60 days. Markers of OS, antioxidant enzymes, monoamine oxidase, nuclear factor (erythroid-derived 2)-like 2 (NRF-2), nitric oxide-2 (NOS-2), inter cellular adhesion molecule-1 (ICAM-1), and neurotrophins are measured. Hyperlipidemia increases OS, decreases antioxidant enzyme activity, increases monoamine oxidase activity, increases NOS-2 and ICAM-1 expression, decreases NRF-2 activation, decreases nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) levels in the brain compared to control. While EPA+DHA and zerumbone significantly (p < 0.05) restores the perturbations induced by hyperlipidemia. CONCLUSION: It is concluded that hyperlipidemia cause OS by decreasing the activity of brain antioxidant enzymes via the downregulation of NRF-2. The reduced brain neurotrophins in hyperlipidemia indicate its potential risk on cognitive attributes. EPA+DHA, together with zerumbone, positively modulates hyperlipidemia induced brain dysfunction thereby offering promising therapeutic strategy.

Zerumbone augments cognitive enhancement potentials of EPA+DHA: insight from a hyperlipidaemic rat model.

Hyperlipidaemia and cognitive dysfunction (CD) are the two public health concerns. Though hyperlipidaemia has been comprehensively studied in respect to CVD, its role on CD needs to be explored. Hence, we evaluated hyperlipidaemia as a risk factor for CD and the efficacy of EPA (20 : 5n-3) + DHA (22 : 6n-3) and zerumbone (Z) in modulating CD under hyperlipidaemic conditions. Male Wistar rats (Rattus norvegicus) were fed control, high-fat (HF), high-fat + fish oil (HF + F), high-fat + zerumbone (HF+Z) and high-fat + fish oil + zerumbone (HF+F+Z) containing diets. After a 30 d feeding trial, memory parameters (Morris water maze, elevated plus maze (transfer latency) and T-maze (spontaneous alteration)) and locomotor skills (open field test and rotarod test) were assessed. Hyperlipidaemia significantly (P < 0·05) reduced memory and motor coordination skills compared with control. However, the administration of EPA + DHA and zerumbone significantly (P < 0·05) restored the hyperlipidaemia-induced loss of memory and motor coordination skills. Collectively, our data imply that hyperlipidaemia causes CD by decreasing memory and motor coordination skills, and administration of EPA + DHA and zerumbone prevents hyperlipidaemia-induced CD. The augmented effect of EPA + DHA, together with zerumbone, discloses a promising strategy for lowering the severity of CD in hyperlipidaemic conditions.

Evidence on n-3 Fatty Acids and Oleic Acid Role in Retinal Inflammation and Microvascular Integrity: Insight from a Hyperlipidemic Rat Model.

Loss of retinal function due to manifestation of chronic inflammation and oxidative stress in hyperglycemia is well addressed. However, the effect of hyperlipidemia on retinal inflammation and microvascular integrity, and the modulatory effects of oxidation-stable oleic acid and long-chain n-3 fatty acids have never been addressed. The objective of this investigation was to assess the retinoprotective effect of oxidation stable oleic acid and oxidation-susceptible EPA + DHA on retinal inflammation and microvascular integrity, under hyperlipidemic conditions. Male Wistar rats were fed with control (7.0% lard), high-fat (35.0% lard), high-fat with fish oil (17.5% fish oil + 17.5% lard), high-fat with olive oil (17.5% olive oil + 17.5% lard), and high-fat with fish oil and olive oil (11.66% fish oil + 11.66% of olive oil + 11.66% of lard) diet for 90 days. Systemic and retinal inflammation, as measured by eicosanoids and cytokines, retinal expression of NF-kB, capillary degeneration, and pericyte loss, were assessed. Hyperlipidemia significantly (p < 0.05) increased the markers of inflammation (PGE2, LTB4, LTC4, IL-1ß, MCP-1, and TNF-α) in serum and retina. Besides, the retinal NF-kB-p65 expression, capillary degeneration, and pericyte loss were significantly (p < 0.05) increased under hyperlipidemic conditions. Dietary incorporation of oleic acid and EPA + DHA significantly (p < 0.05) suppressed hyperlipidemia-induced effects in the retina. In conclusion, hyperlipidemia causes retinal aberrations by compromising the balance in the inflammatory response and microvascular integrity. Dietary incorporation of oleic acid and long-chain n-3 fatty acids prevents hyperlipidemia-induced aberrations in the retina.

Cysteinyl leukotriene receptor antagonism: a promising pharmacological strategy for lowering the severity of arthritis.

BACKGROUND AND AIMS: Though cyclooxygenase inhibitors are employed in rheumatoid arthritis treatment, modulators of leukotrienes are underexplored. We investigated the therapeutic potential of montelukast, a known cysteinyl leukotriene receptor-1 (CysLT1) inhibitor in an experimental rat model of arthritis. METHODS: Arthritis was induced in rats, and montelukast (5 mg/kg body wt.) was administered prophylactically (PAM) and therapeutically (TAM) through oral route. RESULTS AND DISCUSSION: Blood and joint tissue markers of oxidative stress (lipid peroxidation, protein carbonyls, and nitric oxides) were significantly (p < 0.05) reduced in montelukast administered rats. Paw inflammation, RA markers (RF and CRP), eicosanoids (PGE2, LTB4, and LTC4), cytokines (IL-1ß and MCP-1), activity of hydrolytic enzymes (collagenase, elastase, and hyaluronidase), expression of matrix metalloproteinases (MMP), and EP-4 receptor were significantly (p < 0.05) reduced in montelukast administered rats. This study established that leukotriene inhibition through montelukast lowered the severity of arthritis and thus a potential strategy for reducing the severity of arthritis.

Calcium-Activated Chloride Channels in Newly Differentiating Mouse Lens Fiber Cells and Their Role in Volume Regulation.

Purpose: Chloride channels have been proposed to play an important role in the regulation of lens volume. Unfortunately, little information is available about the molecular identity of these channels or how they are regulated in the lens due to the difficulties in isolating mouse fiber cells. Recently, our laboratory has developed a new technique for isolating these cells by using transgenic mouse lenses that lack both Cx50 and Cx46. The purpose of this study was to test the hypothesis that newly differentiating mouse fiber cells express calcium-activated chloride channels (CaCCs) by using this technique. Methods: Differentiating fiber cells were isolated from lenses of double knockout mice that lack both Cx50 and Cx46 by using collagenase. Membrane currents were studied using the whole-cell patch clamp technique. The molecular identity and distribution of CaCCs were investigated using RT-PCR and immunofluorescence. Results: Our electrophysiologic experiments suggest that peripheral fiber cells express a calcium-activated chloride current. The voltage gating properties, calcium sensitivity, and pharmacologic properties of this current resembled those of TMEM16 CaCCs. RT-PCR analysis demonstrated the presence of TMEM16A and TMEM16B transcripts in wild-type and double knockout mouse lenses. Both TMEM16A and TMEM16B proteins were detected in the differentiating epithelial cells and newly elongating fiber cells near the equator of the lens by immunohistochemistry. Conclusions: Our results demonstrate that membrane conductance of peripheral fiber cells contain CaCCs that can be attributed to TMEM16A and TMEM16B. Given their critical role in volume regulation in other tissues, we speculate that these channels play a similar role in the lens.

n-3 Fatty Acids Abrogate Dyslipidemia-Induced Changes in Bile Acid Uptake, Synthesis, and Transport in Young and Aged Dyslipidemic Rats.

In this study, the effect of n-3 fatty acids (FA) [α-linolenic acid (ALA) and eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA)] on the intestinal bile acid (BA) uptake, hepatic BA synthesis, and enterohepatic bile acid transporters (BAT) was assessed in young and aged dyslipidemic rats. Dyslipidemia was induced in young and aged rats by feeding a high-fat (HF) diet. Experimental groups received diets containing canola oil (HF + CNO) and fish oil (HF + FO) as a source of ALA and EPA + DHA, respectively. After 60 days of feeding, intestinal BA uptake and expression of apical sodium-dependent bile acid transporter (Asbt), organic solute transporter-alpha/beta (Osta/b) messenger RNA (mRNA), and hepatic expression of Na+ taurocholate cotransporting polypeptide (Ntcp), bile salt export pump (Bsep), cholesterol 7-α hydroxylase A1 (Cyp7a1), Farnesoid X receptor (Fxr), small heterodimer partner-1 (Shp), liver receptor homolog-1 (Lrh-1), and hepatic nuclear factor-4 alpha (Hnf4a) mRNA were measured. Hepatic 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase activity and total BA in serum, liver, and feces were assessed. The dyslipidemic HF group had: (1) increased intestinal BA uptake and Asbt and Osta/b mRNA expression, (2) increased BA in serum, (3) decreased hepatic expression of Ntcp, Bsep, and Cyp7a1 mRNA, (4) increased activity of HMG-CoA reductase, (5) increased hepatic expression of Fxr and Shp mRNA, (6) decreased hepatic expression of Lrh-1 and Hnf4a mRNA, and (7) decreased BA in feces, when compared to control, HF + CNO, and HF + FO groups. Immunostaining revealed increased expression of intestinal Asbt and hepatic Ntcp protein in the HF group when compared to control, HF + CNO, and HF + FO groups. n-3 FA abrogated dyslipidemia-induced changes in the intestinal uptake, hepatic synthesis, and enterohepatic transporters of BA in both young and aged rats. EPA + DHA was more effective than ALA in modulating dyslipidemia-induced changes.

Aging and Hyperglycemia Intensify Dyslipidemia-Induced Oxidative Stress and Inflammation in Rats: Assessment of Restorative Potentials of ALA and EPA + DHA.

Effect of aging and hyperglycemia on oxidative stress (OS) and inflammation in dyslipidemic conditions has not been elucidated. Hence, in this study, we assessed the implications of aging, hyperglycemia, and also the dietary effect of n-3 fatty acids (α-linolenic acid (ALA) and eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA)) on OS and inflammation in dyslipidemic rats. Dyslipidemia was induced in young and aged rats by feeding high-fat lard (HFL) diet. Diabetes was induced in young dyslipidemic rats by administering streptozotocin 30 days after the induction of dyslipidemia. Experimental groups received diets containing canola oil (HF + CNO) and fish oil (HF + FO) as a source of ALA and EPA + DHA respectively. After 60 days of feeding rats with their respective diets, OS and inflammatory markers in serum were assessed. Dyslipidemia caused significant (p < 0.05) increase in OS (lipid peroxidation, nitric oxide, and protein carbonyl), pro-inflammatory cytokine (CRP, IL-1ß, MCP-1, and TNF-α), and eicosanoid (PGE2, LTB4, and LTC4) level in serum of both young and aged rats. Aged dyslipidemic rats presented significantly (p < 0.05) higher level of these markers compared to young dyslipidemic rats. Hyperglycemia onset further augmented OS and inflammatory markers in young dyslipidemic rats significantly (p < 0.05). Administration of n-3 fatty acids downregulated the serum markers of OS and inflammation in all the three experimental models. Thus, aging and hyperglycemia onset intensified dyslipidemia-induced OS and inflammation. Dietary preformed EPA + DHA presented larger restorative potentials than precursor ALA in countering OS and inflammation in all the three experimental models.

Sesame Oil and Rice Bran Oil Ameliorates Adjuvant-Induced Arthritis in Rats: Distinguishing the Role of Minor Components and Fatty Acids.

Though present in small amounts, the minor constituents of dietary oils may supplement the dietary therapies for rheumatoid arthritis (RA). Hence, in the present study, we assessed the effect of minor constituents from sesame oil (SO) and rice bran oil (RBO) and their fatty acids on the severity of adjuvant-induced arthritis in experimental rats. Rats were gavaged with 1 mL of SO or RBO or groundnut oil (GNO, control) with or without its minor components for a period 15 days before and 15 days after the induction of arthritis. Oxidative stress, markers of RA, eicosanoids, cytokines, paw swelling and joint integrity were measured in experimental and control rats. Results demonstrated that native SO and RBO but not SO and RBO stripped of their minor components decreased severity of paw inflammation, oxidative stress (lipid peroxides, protein carbonyls, nitric oxide), RA markers (RF and CRP), inflammatory eicosanoids (PGE2, LTB4 and LTC4) and cytokines (IL-1ß, IL-6, MCP-1 and TNF-α) compared to control rats. Native SO and RBO inhibited hydrolytic enzymes (collagenase, elastase and hyaluronidase) in the synovial tissue compared to SO and RBO without minor components. The arthritic scores assessed based on the digital and X-ray images indicated that native oils but not those without their minor components reduced the paw swelling and bone loss. Our results indicated that minor components of SO and RBO possess a significant degree of an anti-arthritic effect and are responsible for down regulating inflammation in the experimentally induced arthritis in rats.