The role of phosphorus speciation of biochar in reducing available Cd and phytoavailability in mining area soil: Effect and mechanism.

The effect of phosphorus (P) speciation in biochar on soil available Cd and its mechanism to alleviate plant Cd stress remain largely unknown. Here, ammonium polyphosphate (PABC)-, phosphoric acid (PHBC)-, potassium dihydrogen phosphate (PKBC)-, and ammonium dihydrogen phosphate (PNBC)-modified biochar were used to investigate P speciation. The Cd immobilization mechanism of biochar was analyzed by XPS and 31P NMR, and the soil quality and the mechanism for the biochar to alleviate Cd stress were also determined. The results demonstrated that PBC (pristine biochar), PABC, PHBC, PKBC, and PNBC reduced the content of soil DTPA-Cd by 14.96 % - 32.19 %, 40.44 % - 47.26 %, 17.52 % - 41.78 %, and 21.90 % - 36.64 %, respectively. The XPS and 31P NMR results demonstrated that the orthophosphate on the surface of PABC, PHBC, PKBC, and PNBC accounted for 82.06 %, 62.77 %, 33.1 %, and 54.46 %, respectively, indicating that PABC has the highest passivation efficiency on soil Cd, which was ascribed to the highest orthophosphate content on the biochar surface. Pot experiments revealed that PABC could reduce the Cd content by 4.18, 4.41, 4.43, 2.94, and 2.57 folds in roots, stems, leaves, pods, and grains, respectively, and at the same time increase the dry and fresh weight of soybean and decrease Cd toxicity to soybean by improving the antioxidant system. In addition, application of the P-modified biochars improved the enzyme activity and physicochemical properties of the soil. This study provides a new perspective for studying the effect of P-modified biochars on soil Cd immobilization.

Intestinal activating transcription factor 4 regulates stress-related behavioral alterations via paraventricular thalamus in male mice.

Chronic stress induces depression- and anxiety-related behaviors, which are common mental disorders accompanied not only by dysfunction of the brain but also of the intestine. Activating transcription factor 4 (ATF4) is a stress-induced gene, and we previously show that it is important for gut functions; however, the contribution of the intestinal ATF4 to stress-related behaviors is not known. Here, we show that chronic stress inhibits the expression of ATF4 in gut epithelial cells. ATF4 overexpression in the colon relieves stress-related behavioral alterations in male mice, as measured by open-field test, elevated plus-maze test, and tail suspension test, whereas intestine-specific ATF4 knockout induces stress-related behavioral alterations in male mice. Furthermore, glutamatergic neurons are inhibited in the paraventricular thalamus (PVT) of two strains of intestinal ATF4-deficient mice, and selective activation of these neurons alleviates stress-related behavioral alterations in intestinal ATF4-deficient mice. The highly expressed gut-secreted peptide trefoil factor 3 (TFF3) is chosen from RNA-Seq data from ATF4 deletion mice and demonstrated decreased in gut epithelial cells, which is directly regulated by ATF4. Injection of TFF3 reverses stress-related behaviors in ATF4 knockout mice, and the beneficial effects of TFF3 are blocked by inhibiting PVT glutamatergic neurons using DREADDs. In summary, this study demonstrates the function of ATF4 in the gut-brain regulation of stress-related behavioral alterations, via TFF3 modulating PVT neural activity. This research provides evidence of gut signals regulating stress-related behavioral alterations and identifies possible drug targets for the treatment of stress-related behavioral disorders.

Inhibition of c-Jun in AgRP neurons increases stress-induced anxiety and colitis susceptibility.

Psychiatric disorders, such as anxiety, are associated with inflammatory bowel disease (IBD), however, the neural mechanisms regulating this comorbidity are unknown. Here, we show that hypothalamic agouti-related protein (AgRP) neuronal activity is suppressed under chronic restraint stress (CRS), a condition known to increase anxiety and colitis susceptibility. Consistently, chemogenic activation or inhibition of AgRP neurons reverses or mimics CRS-induced increase of anxiety-like behaviors and colitis susceptibility, respectively. Furthermore, CRS inhibits AgRP neuronal activity by suppressing the expression of c-Jun. Moreover, overexpression of c-Jun in these neurons protects against the CRS-induced effects, and knockdown of c-Jun in AgRP neurons (c-Jun∆AgRP) promotes anxiety and colitis susceptibility. Finally, the levels of secreted protein thrombospondin 1 (THBS1) are negatively associated with increased anxiety and colitis, and supplementing recombinant THBS1 rescues colitis susceptibility in c-Jun∆AgRP mice. Taken together, these results reveal critical roles of hypothalamic AgRP neuron-derived c-Jun in orchestrating stress-induced anxiety and colitis susceptibility.

Uncovering the pharmacological mechanisms of Zizhu ointment against diabetic ulcer by integrating network analysis and experimental evaluation in vivo and in vitro.

Diabetic ulcer (DU) has been recognized as one of the most prevalent and serious complications of diabetes. However, the clinical efficacy of standard treatments for DU remains poor. Traditional Chinese medicine (TCM) shows a positive therapeutic effect on DU. Specifically, Zizhu ointment (ZZO) has been widely used to treat DU in long-term clinical practice, but the exact mechanism by which it promotes DU wound healing remains unknown. In this study, network analysis and high-performance liquid chromatography-high resolution mass spectrometry (UPLC-HRMS) were conducted to identify the active compounds of ZZO. We detected isovalerylshikonin (ISO), mandenol, daidzein, kaempferol, and formononetin in both network analysis and UPLC-HRMS. Moreover, ZZO could ameliorate DU by regulating the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) and inflammation signaling pathways, according to the results of KEGG analysis. We established a DU mouse model with a high-fat diet and streptozotocin injection in vivo to evaluate the network analysis result. The experimental results showed that ZZO could inhibit inflammation, remodel fibrous tissue, and promote angiogenesis in the DU area, facilitating wound healing in DU mice. Moreover, the PI3K/AKT signaling pathway was indeed activated by ZZO treatment, promoting macrophage M2 polarization. In addition, we used molecular docking technology to evaluate the binding sites between ZZO and the PI3K/AKT pathway. The results showed that ISO has a good binding interaction with AKT. Moreover, ISO promoted M2 polarization in macrophages in a dose-dependent manner in vitro. Our study found that ZZO could promote DU wound healing by inhibiting inflammation, which was achieved by macrophage M2 polarization through activating the PI3K/AKT pathway. Further studies have demonstrated that ISO plays major role in the above process. These findings provide a theoretical basis for further preclinical evaluation and lay a foundation for nano-gel compound treatment with ZZO.

Chromosome-level genome of Camellia lanceoleosa provides a valuable resource for understanding genome evolution and self-incompatibility.

The section Oleifera (Theaceae) has attracted attention for the high levels of unsaturated fatty acids found in its seeds. Here, we report the chromosome-scale genome of the sect. Oleifera using diploid wild Camellia lanceoleosa with a final size of 3.00 Gb and an N50 scaffold size of 186.43 Mb. Repetitive sequences accounted for 80.63% and were distributed unevenly across the genome. Camellia lanceoleosa underwent a whole-genome duplication event approximately 65 million years ago (65 Mya), prior to the divergence of C. lanceoleosa and Camellia sinensis (approx. 6-7 Mya). Syntenic comparisons of these two species elucidated the genomic rearrangement, appearing to be driven in part by the activity of transposable elements. The expanded and positively selected genes in C. lanceoleosa were significantly enriched in oil biosynthesis, and the expansion of homomeric acetyl-coenzyme A carboxylase (ACCase) genes and the seed-biased expression of genes encoding heteromeric ACCase, diacylglycerol acyltransferase, glyceraldehyde-3-phosphate dehydrogenase and stearoyl-ACP desaturase could be of primary importance for the high oil and oleic acid content found in C. lanceoleosa. Theanine and catechins were present in the leaves of C. lanceoleosa. However, caffeine can not be dectected in the leaves but was abundant in the seeds and roots. The functional and transcriptional divergence of genes encoding SAM-dependent N-methyltransferases may be associated with caffeine accumulation and distribution. Gene expression profiles, structural composition and chromosomal location suggest that the late-acting self-incompatibility of C. lanceoleosa is likely to have favoured a novel mechanism co-occurring with gametophytic self-incompatibility. This study provides valuable resources for quantitative and qualitative improvements and genome assembly of polyploid plants in sect. Oleifera.

Rational Design of All-Organic Nanoplatform for Highly Efficient MR/NIR-II Imaging-Guided Cancer Phototheranostics.

Organic theranostic nanomedicine has precision multimodel imaging capability and concurrent therapeutics under noninvasive imaging guidance. However, the rational design of desirable multifunctional organic theranostics for cancer remains challenging. Rational engineering of organic semiconducting nanomaterials has revealed great potential for cancer theranostics largely owing to their intrinsic diversified biophotonics, easy fabrication of multimodel imaging platform, and desirable biocompatibility. Herein, a novel all-organic nanotheranostic platform (TPATQ-PNP NPs) is developed by exploiting the self-assembly of a semiconducting small molecule (TPATQ) and a new synthetic high-density nitroxide radical-based amphiphilic polymer (PNP). The nitroxide radicals act as metal-free magnetic resonance imaging agent through shortened longitudinal relaxation times, and the semiconducting molecules enable ultralow background second near-infrared (NIR-II, 1000-1700 nm) fluorescence imaging. The as-prepared TPATQ-PNP NPs can light up whole blood vessels of mice and show precision tumor-locating ability with synergistic (MR/NIR-II) imaging modalities. The semiconducting molecules also undergo highly effective photothermal conversion in the NIR region for cancer photothermal therapy guided by complementary tumor diagnosis. The designed multifunctional organic semiconducting self-assembly provides new insights into the development of a new platform for cancer theranostics.

Rehmannia Radix Extract Relieves Bleomycin-Induced Pulmonary Fibrosis in Mice via Transforming Growth Factor ß1 (TGF-ß1).

BACKGROUND Infants and young children with acute respiratory distress syndrome (ARDS) have acute progressive hypoxic respiratory failure caused by a variety of extrapulmonary pathogenic factors and cardiogenic factors. Diffuse alveolar injury and pulmonary fibrosis both are pathological features of ARDS. This study investigated the effect of Rehmannia Radix extract (RRE) on pulmonary fibrosis of infants with ARDS. MATERIAL AND METHODS The human lung fibroblasts cell line HFL1 was treated with various concentrations of Rehmannia Radix extract in different groups for different times. Flow cytometry and TUNEL assay were performed to detect cell apoptosis, and CCK8 assay was utilized to analyze cell proliferation. TGF-ß1 expression was detected by real-time quantitative PCR, and protein-level expressions of Caspase3, TGF-ß1, Bcl-2, and Smad3 were measured by western blot and immunohistochemical staining in cells or tissues. TGF-ß1 was overexpressed by recombinant human TGF-ß1 (2 ng/mL) and the treated cells and culture supernatant were harvested for analysis in each step. Bleomycin was used to induce a mouse model of pulmonary fibrosis and was confirmed by HE pathological sections. RESULTS Flow cytometry and TUNEL results showed that RRE promoted the apoptosis of HFL1 cells in a concentration-dependent manner, and it inhibited the proliferation of HFL1 cells. Upregulation of TGF-ß1 reversed the effects of RRE in HFL1 cells. RRE alleviated pulmonary fibrosis in mice through downregulating Bcl-2, TGF-ß1, and Smad3 expression. CONCLUSIONS RRE promoted apoptosis and inhibited proliferation of HFL1, and then arrested the progression of pulmonary fibrosis. RRE had a significant inhibitory effect on TGF-ß1 and Smad3. These results suggest that RRE directly prevents the development of pulmonary fibrosis by affecting the expression of TGF-ß1 and Smad3.

Gadolinium-Chelated Conjugated Polymer-Based Nanotheranostics for Photoacoustic/Magnetic Resonance/NIR-II Fluorescence Imaging-Guided Cancer Photothermal Therapy.

Our exploiting versatile multimodal theranostic agent aims to integrate the complementary superiorities of photoacoustic imaging (PAI), second near-infrared (NIR-II, 1000-1700) fluorescence and T1-weighted magnetic resonance imaging (MRI) with an ultimate objective of perfecting cancer diagnosis, thus improving cancer therapy efficacy. Herein, we engineered and prepared a water-soluble gadolinium-chelated conjugated polymer-based theranostic nanomedicine (PFTQ-PEG-Gd NPs) for in vivo tri-mode PA/MR/NIR-II imaging-guided tumor photothermal therapy (PTT). Methods: We firstly constructed a semiconducting polymer composed of low-bandgap donor-acceptor (D-A) which afforded the strong NIR absorption for PAI/PTT and long fluorescence emission to NIR-II region for in vivo imaging. Then, the remaining carboxyl groups of the polymeric NPs could effectively chelate with Gd3+ ions for MRI. The in vitro characteristics of the PFTQ-PEG-Gd NPs were studied and the in vivo multimode imaging as well as anti-tumor efficacy of the NPs was evaluated using 4T1 tumor-bearing mice. Results: The obtained theranostic agent showed excellent chemical and optical stability as well as low biotoxicity. After 24 h of systemic administration using PQTF-PEG-Gd NPs, the tumor sites of living mice exhibited obvious enhancement in PA, NIR-II fluorescence and positive MR signal intensities. Better still, a conspicuous tumor growth restraint was detected under NIR light irradiation after administration of PQTF-PEG-Gd NPs, indicating the efficient photothermal potency of the nano-agent. Conclusion: we triumphantly designed and synthesized a novel and omnipotent semiconducting polymer nanoparticles-based theranostic platform for PAI, NIR-II fluorescence imaging as well as positive MRI-guided tumor PTT in living mice. We expect that such a novel organic nano-platform manifests a great promise for high spatial resolution and deep penetration cancer theranostics.

Semiconducting polymer nanotheranostics for NIR-II/Photoacoustic imaging-guided photothermal initiated nitric oxide/photothermal therapy.

Gasotransmitters with their cytotoxicity in high concentration have become the focus of attention. For such concentration depended therapy, how to effectively deliver gases and precisely control gases release to the lesion as well as combine them with other therapy to achieve precise therapeutics is still a big challenge. Herein, we realize single near-infrared (NIR) laser-initiated nitric oxide (NO) therapy/photothermal therapy (PTT) using semiconducting polymer nanoparticles (SPNs, PFTDPP) combing s-nitrosothiol groups (the NO donor, SNAP). By the good photothermal conversion effect of SPNs, NIR laser energy can be spatio-temporally controlled to convert into heat to decompose s-nitrosothiol. Meanwhile, considering the accompanied PTT produced by photothermal, we can easily and precisely conduct a dual therapy (NO therapy/PTT) under single NIR laser irradiation. Additionally, semiconducting polymer with its structural modifiability and spectral adjustability can provide a second NIR window & photoacoustic (NIR II/PA) imaging for guiding photothermal initiated NO/photothermal therapy. PFTDPP showed a high photothermal conversion efficiency of 48% and good dual-mode imaging signals (NIR-II/photoacoustic). Cellular test illustrated that NO combined photothermal presented more prominent cytotoxicity than any one of them individually. As the tumor pinpointed in vivo by dual-mode imaging (NIR II/PA), this nanotheranostics provided a tumor inhibition of 77%. Consequently, such phototheranostics produced a new design thought for effectively deliver and precisely controlled release of drugs for oncology. And also, it expanded the application range of gasotransmitters combined therapy that shall have a promising application foreground.

ATF4 Deficiency Promotes Intestinal Inflammation in Mice by Reducing Uptake of Glutamine and Expression of Antimicrobial Peptides.

BACKGROUND & AIMS: Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis. METHODS: We obtained biopsy samples during endoscopy from inflamed and/or uninflamed regions of the colon from 21 patients with active Crohn's disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4ΔIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin α1 (DEFA1) and supplementation of l-alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by high-performance liquid chromatography-tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing. RESULTS: Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4ΔIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4ΔIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4ΔIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4ΔIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4. CONCLUSIONS: Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.

Post-stroke administration of omega-3 polyunsaturated fatty acids promotes neurovascular restoration after ischemic stroke in mice: Efficacy declines with aging.

Post-stroke treatment with omega-3 polyunsaturated fatty acids (n-3 PUFAs) may be a promising therapy in young animals but this has not been tested in aged subjects, a population at most risk of ischemic stroke. Herein we examined the therapeutic efficacy of n-3 PUFAs after distal middle cerebral artery occlusion (dMCAO) in young (10-12 weeks old) and aged (18 months old) mice. Post-ischemic mice were randomly assigned to 4 groups that received: 1) regular food with low content of n-3 PUFAs, 2) intraperitoneal docosahexaenoic acid (DHA, a major component of n-3 PUFAs) injections, 3) Fish oil (FO, containing high concentration of n-3 PUFAs) dietary supplement, or 4) combined treatment with DHA and FO dietary supplement. Long-term neurorestoration induced by n-3 PUFA post-stroke administration and its underlying mechanism(s) were analyzed up to 35 days after dMCAO. Aged mice showed more severe neurological deficits than young mice after dMCAO with histological lesions extended to the striatum. Notably, post-stroke treatment with combined DHA injections and FO dietary supplementation was more effective in reducing brain injury and improving sensorimotor function in aged mice than either treatment alone, albeit to a lesser extent than in the young mice. Unlike the improvement in spatial cognitive function observed in young mice, the combined treatment regimen failed to improve cognitive function in aged mice. The reduction in stroke-induced neurological deficits with n-3 PUFA post-treatment was associated with enhanced angiogenesis, oligodendrogenesis, neuron survival and white matter restoration. Together, these results indicate that the neurological benefits of n-3 PUFA administration after stroke extend to older animals and are associated with improved neuronal survival and brain remodeling, therefore suggesting that post-stroke administration of n-3 PUFAs is a viable clinically relevant treatment option against stroke.

A Post-stroke Therapeutic Regimen with Omega-3 Polyunsaturated Fatty Acids that Promotes White Matter Integrity and Beneficial Microglial Responses after Cerebral Ischemia.

White matter injury induced by ischemic stroke elicits sensorimotor impairments, which can be further deteriorated by persistent proinflammatory responses. We previously reported that delayed and repeated treatments with omega-3 polyunsaturated fatty acids (n-3 PUFAs) improve spatial cognitive functions and hippocampal integrity after ischemic stroke. In the present study, we report a post-stroke n-3 PUFA therapeutic regimen that not only confers protection against neuronal loss in the gray matter but also promotes white matter integrity. Beginning 2 h after 60 min of middle cerebral artery occlusion (MCAO), mice were randomly assigned to receive intraperitoneal docosahexaenoic acid (DHA) injections (10 mg/kg, daily for 14 days), alone or in combination with dietary fish oil (FO) supplements starting 5 days after MCAO. Sensorimotor functions, gray and white matter injury, and microglial responses were examined up to 28 days after MCAO. Our results showed that DHA and FO combined treatment-facilitated long-term sensorimotor recovery and demonstrated greater beneficial effect than DHA injections alone. Mechanistically, n-3 PUFAs not only offered direct protection on white matter components, such as oligodendrocytes, but also potentiated microglial M2 polarization, which may be important for white matter repair. Notably, the improved white matter integrity and increased M2 microglia were strongly linked to the mitigation of sensorimotor deficits after stroke upon n-3 PUFA treatments. Together, our results suggest that post-stroke DHA injections in combination with FO dietary supplement benefit white matter restoration and microglial responses, thereby dictating long-term functional improvements.

Delayed Docosahexaenoic Acid Treatment Combined with Dietary Supplementation of Omega-3 Fatty Acids Promotes Long-Term Neurovascular Restoration After Ischemic Stroke.

Prophylactic dietary intake of omega-3 polyunsaturated fatty acids (n-3 PUFAs) has been shown to remarkably ameliorate ischemic brain injury. However, the therapeutic efficacy of n-3 PUFA administration post-stroke, especially its impact on neurovascular remodeling and long-term neurological recovery, has not been fully characterized thus far. In this study, we investigated the effect of n-3 PUFA supplementation, as well as in combination with docosahexaenoic acid (DHA) injections, on long-term stroke outcomes. Mice were subjected to transient middle cerebral artery occlusion (MCAO) before randomly assigned to four groups to receive the following: (1) low dose of n-3 PUFAs as the vehicle control, (2) intraperitoneal DHA injections, (3) n-3 PUFA dietary supplement, or (4) combined treatment of (2) and (3). Neurological deficits and brain atrophy, neurogenesis, angiogenesis, and glial scar formation were assessed up to 28 days after MCAO. Results revealed that groups 2 and 3 showed only marginal reduction in post-stroke tissue loss and attenuation of cognitive deficits. Interestingly, group 4 exhibited significantly reduced tissue atrophy and improved cognitive functions compared to groups 2 and 3 with just a single treatment. Mechanistically, the combined treatment promoted post-stroke neurogenesis and angiogenesis, as well as reduced glial scar formation, all of which significantly correlated with the improved spatial memory in the Morris water maze. These results demonstrate an effective therapeutic regimen to enhance neurovascular restoration and long-term cognitive recovery in the mouse model of MCAO. Combined post-stroke DHA treatment and n-3 PUFA dietary supplementation thus may be a potential clinically translatable therapy for stroke or related brain disorders.

Omega-3 polyunsaturated fatty acids mitigate blood-brain barrier disruption after hypoxic-ischemic brain injury.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) have been shown to protect the neonatal brain against hypoxic/ischemic (H/I) injury. However, the mechanism of n-3 PUFA-afforded neuroprotection is not well understood. One major determinant of H/I vulnerability is the permeability of the blood-brain barrier (BBB). Therefore, we examined the effects of n-3 PUFAs on BBB integrity after neonatal H/I. Female rats were fed a diet with or without n-3 PUFA enrichment from day 2 of pregnancy to 14days after parturition. H/I was introduced in 7day-old offspring. We observed relatively rapid BBB penetration of the small molecule cadaverine (640Da) at 4h post-H/I and a delayed penetration of larger dextrans (3kD-40kD) 24-48h after injury. Surprisingly, the neonatal BBB was impermeable to Evans Blue or 70kD dextran leakage for up to 48h post-H/I, despite evidence of IgG extravasation at this time. As expected, n-3 PUFAs ameliorated H/I-induced BBB damage, as shown by reductions in tracer efflux and IgG extravasation, preservation of BBB ultrastructure, and enhanced tight junction protein expression. Furthermore, n-3 PUFAs prevented the elevation in matrix metalloproteinase (MMP) activity in the brain and blood after H/I. Thus, n-3 PUFAs may protect neonates against BBB damage by blunting MMPs activation after H/I.

n-3 Polyunsaturated Fatty Acids Reduce Neonatal Hypoxic/Ischemic Brain Injury by Promoting Phosphatidylserine Formation and Akt Signaling.

BACKGROUND AND PURPOSE: Omega-3 polyunsaturated fatty acids (n-3 PUFAs) attenuate neonatal hypoxic/ischemic (H/I) brain damage, but the underlying mechanisms are not fully understood. This study tested the hypothesis that n-3 PUFAs enhance Akt-dependent prosurvival signaling by promoting the biosynthesis of phosphatidylserine in neuronal cell membranes. METHODS: Dietary n-3 PUFA supplementation was initiated on the second day of pregnancy in dams. H/I was induced in 7-day-old rat pups by ipsilateral common carotid artery occlusion followed by hypoxia (8% oxygen for 2.5 hours). Neurological outcomes, brain tissue loss, cell death, and the activation of signaling events were assessed after H/I. The effects of n-3 PUFAs (docosahexaenoic acid and eicosapentaenoic acid) on oxygen-glucose deprivation-induced cell death and the underlying mechanism of protection were also examined in primary cortical neuron cultures. RESULTS: n-3 PUFAs reduced brain tissue loss at 7 days after H/I and improved neurological outcomes, whereas inhibition of PI3K/Akt signaling by LY294002 partially abrogated this neuroprotective effect. Docosahexaenoic acid/eicosapentaenoic acid also prevented ischemic neuronal death through the Akt prosurvival pathway in vitro. Furthermore, docosahexaenoic acid/eicosapentaenoic acid increased the production of phosphatidylserine, the major membrane-bound phospholipids, after ischemia both in vitro and in vivo. A reduction in membrane phosphatidylserine by shRNA-mediated knockdown of phosphatidylserine synthetase-1 attenuated Akt activation and neuronal survival after docosahexaenoic acid/eicosapentaenoic acid treatment in the oxygen-glucose deprivation model. CONCLUSIONS: n-3 PUFAs robustly protect against H/I-induced brain damage in neonates by activating Akt prosurvival pathway in compromised neurons. In addition, n-3 PUFAs promote the formation of membrane phosphatidylserine, thereby promoting Akt activity and improving cellular survival.

Dietary supplementation with omega-3 polyunsaturated fatty acids robustly promotes neurovascular restorative dynamics and improves neurological functions after stroke.

Stroke is a devastating neurological disease with no satisfactory therapies to preserve long-term neurological function, perhaps due to the sole emphasis on neuronal survival in most preclinical studies. Recent studies have revealed the importance of protecting multiple cell types in the injured brain, such as oligodendrocytes and components of the neurovascular unit, before long-lasting recovery of function can be achieved. For example, revascularization in the ischemic penumbra is critical to provide various neurotrophic factors that enhance the survival and activity of neurons and other progenitor cells, such as oligodendrocyte precursor cells. In the present study, we hypothesized that chronic dietary supplementation with fish oil promotes post-stroke angiogenesis, neurogenesis, and oligodendrogenesis, thereby leading to long-term functional improvements. Mice received dietary supplementation with n-3 PUFA-enriched fish oil for three months before and up to one month after stroke. As expected, dietary n-3 PUFAs significantly increased levels of n-3 PUFAs in the brain and improved long-term behavioral outcomes after cerebral ischemia. n-3 PUFAs also robustly improved revascularization and angiogenesis and boosted the survival of NeuN/BrdU labeled newborn neurons up to 35days after stroke injury. Furthermore, these pro-neurogenic effects were accompanied by robust oligodendrogenesis. Thus, this is the first study to demonstrate that chronic dietary intake of n-3 PUFAs is an effective prophylactic measure not only to protect against ischemic injury for the long term but also to actively promote neurovascular restorative dynamics and brain repair.

n-3 PUFA supplementation benefits microglial responses to myelin pathology.

Microglia represent rational but challenging targets for improving white matter integrity because of their dualistic protective and toxic roles. The present study examines the effect of Omega-3 polyunsaturated fatty acids (n-3 PUFAs) on microglial responses to myelin pathology in primary cultures and in the cuprizone mouse model of multiple sclerosis (MS), a devastating demyelination disease. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), the two main forms of n-3 PUFAs in the brain, inhibited the release of nitric oxide and tumor necrosis factor-α from primary microglia upon IFN-γ and myelin stimulation. DHA and EPA also enhanced myelin phagocytosis in vitro. Therefore, n-3 PUFAs can inhibit inflammation while at the same time enhancing beneficial immune responses such as microglial phagocytosis. In vivo studies demonstrated that n-3 PUFA supplementation reduced cuprizone-induced demyelination and improved motor and cognitive function. The positive effects of n-3 PUFAs were accompanied by a shift in microglial polarization toward the beneficial M2 phenotype both in vitro and in vivo. These results suggest that n-3 PUFAs may be clinically useful as immunomodulatory agents for demyelinating diseases through a novel mechanism involving microglial phenotype switching.

Omega-3 polyunsaturated fatty acids enhance cerebral angiogenesis and provide long-term protection after stroke.

Stroke is a devastating neurological disorder and one of the leading causes of death and serious disability. After cerebral ischemia, revascularization in the ischemic boundary zone provides nutritive blood flow as well as various growth factors to promote the survival and activity of neurons and neural progenitor cells. Enhancement of angiogenesis and the resulting improvement of cerebral microcirculation are key restorative mechanisms and represent an important therapeutic strategy for ischemic stroke. In the present study, we tested the hypothesis that post-stroke angiogenesis would be enhanced by omega-3 polyunsaturated fatty acids (n-3 PUFAs), a major component of dietary fish oil. To this end, we found that transgenic fat-1 mice that overproduce n-3 PUFAs exhibited long-term behavioral and histological protection against transient focal cerebral ischemia (tFCI). Importantly, fat-1 transgenic mice also exhibited robust improvements in revascularization and angiogenesis compared to wild type littermates, suggesting a potential role for n-3 fatty acids in post-stroke cerebrovascular remodeling. Mechanistically, n-3 PUFAs induced upregulation of angiopoietin 2 (Ang 2) in astrocytes after tFCI and stimulated extracellular Ang 2 release from cultured astrocytes after oxygen and glucose deprivation. Ang 2 facilitated endothelial proliferation and barrier formation in vitro by potentiating the effects of VEGF on phospholipase Cγ1 and Src signaling. Consistent with these findings, blockade of Src activity in post-stroke fat-1 mice impaired n-3 PUFA-induced angiogenesis and exacerbated long-term neurological outcomes. Taken together, our findings strongly suggest that n-3 PUFA supplementation is a potential angiogenic treatment capable of augmenting brain repair and improving long-term functional recovery after cerebral ischemia.

Omega-3 fatty acids protect the brain against ischemic injury by activating Nrf2 and upregulating heme oxygenase 1.

Ischemic stroke is a debilitating clinical disorder that affects millions of people, yet lacks effective neuroprotective treatments. Fish oil is known to exert beneficial effects against cerebral ischemia. However, the underlying protective mechanisms are not fully understood. The present study tests the hypothesis that omega-3 polyunsaturated fatty acids (n-3 PUFAs) attenuate ischemic neuronal injury by activating nuclear factor E2-related factor 2 (Nrf2) and upregulating heme oxygenase-1 (HO-1) in both in vitro and in vivo models. We observed that pretreatment of rat primary neurons with docosahexaenoic acid (DHA) significantly reduced neuronal death following oxygen-glucose deprivation. This protection was associated with increased Nrf2 activation and HO-1 upregulation. Inhibition of HO-1 activity with tin protoporphyrin IX attenuated the protective effects of DHA. Further studies showed that 4-hydroxy-2E-hexenal (4-HHE), an end-product of peroxidation of n-3 PUFAs, was a more potent Nrf2 inducer than 4-hydroxy-2E-nonenal derived from n-6 PUFAs. In an in vivo setting, transgenic mice overexpressing fatty acid metabolism-1, an enzyme that converts n-6 PUFAs to n-3 PUFAs, were remarkably resistant to focal cerebral ischemia compared with their wild-type littermates. Regular mice fed with a fish oil-enhanced diet also demonstrated significant resistance to ischemia compared with mice fed with a regular diet. As expected, the protection was associated with HO-1 upregulation, Nrf2 activation, and 4-HHE generation. Together, our data demonstrate that n-3 PUFAs are highly effective in protecting the brain, and that the protective mechanisms involve Nrf2 activation and HO-1 upregulation by 4-HHE. Further investigation of n-3 PUFA neuroprotective mechanisms may accelerate the development of stroke therapies.

Omega-3 polyunsaturated fatty acids in the brain: metabolism and neuroprotection.

Omega-3 polyunsaturated fatty acids (n-3 PUFAs) are a group of essential fatty acids that serve as energy substrates and integral membrane components, and therefore play crucial roles in the maintenance of normal neurological function. Recent studies show that n-3 PUFAs display neuroprotective properties and exert beneficial effects on the cognitive function with aging. The brain's need of n-3 PUFAs is predominantly met by the blood delivery due to their limited synthesis in the brain. The present review focuses on the metabolism of n-3 PUFAs in the brain, including their accumulation and turnover. We also highlight the current understanding of the neuroprotective effects of n-3 PUFAs against cerebral ischemia and neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease.