Inhibitory effect of astaxanthin on oxidative stress damage in lens epithelial cells and its mechanism / 中华实验眼科杂志

Objective:To investigate the regulatory effect of astaxanthin on oxidative stress injury induced by hydrogen peroxide (H 2O 2) in lens epithelial cells and its possible mechanism. Methods:The HLEB-3 cells were cultured with different concentrations (0, 50, 100, 200, 500, 750 μmol/L) of H 2O 2.The cell inhibition rate was detected by the methyl thiazolyl tetrazolium (MTT) method, and the 50%inhibiting concentration (IC50) was calculated.HLEB-3 cells were cultured with different concentrations (0, 5, 10, 20, 50 μmol/L) of astaxanthin.The cell survival rate was detected by the MTT method.HLEB-3 cells were divided into four groups for 24-hour culture, namely normal control group cultured with complete medium, oxidative stress group cultured with 250 μmol/L H 2O 2, 10 μmol/L astaxanthin group cultured with 10 μmol/L astaxanthin and 250 μmol/L H 2O 2, and 20 μmol/L astaxanthin group cultured with 20 μmol/L astaxanthin and 250 μmol/L H 2O 2.The cell apoptosis rate was determined by flow cytometry.The nitric oxide (NO) concentration, superoxide dismutase (SOD) activity, glutathione (GSH) activity and malondialdehyde (MDA) content were detected by ELISA.The protein expressions of nuclear factor erythroid-2 related factor 2 (Nrf2) in nuclei, cytoplasmic Nrf2, heme oxygenase-1 (HO-1) and NAD (P) H, quinine oxidoreductase 1 (NQO1) were detected by Western bolt.The cells were divided into four groups, namely normal control-small interfering RNA (NC-siRNA) group, Nrf2-siRNA group, NC-siRNA+ astaxanthin group and Nrf2-siRNA+ astaxanthin group.The cells were transfected with NC-siRNA or Nrf2-siRNA accordingly.The cells were co-cultured for 24 hours with 0/10 μmol/L astaxanthin and 250 μmol/L H 2O 2 24 hours after transfection, respectively.The cell apoptosis rate was determined by flow cytometry.The NO concentration, SOD activity, GSH activity and MDA content were detected by ELISA. Results:With the increase of H 2O 2 concentration, the inhibition rate of HLEB-3 cells increased.There were significant differences in the inhibition rate of HLEB-3 cells treated with different concentrations of H 2O 2 ( F=12.358, P<0.05). The IC50 value of H 2O 2 on HLEB-3 cells was 264.20 μmol/L.The survival rates of HLEB-3 cells treated with 0, 5, 10, 20 and 50 μmol/L astaxanthin were (100.00±0.00)%, (102.20±1.34)%, (109.50±3.60)%, (115.40±4.13)%, (93.60±2.59)%, respectively.Then 10 μmol/L and 20 μmol/L were chosen as the experimental dose.The cell apoptosis rate of oxidative stress group was (38.50±2.38)%, which was higher than (9.20±0.24)% of normal control group, with a statistically significant difference ( P<0.05). The cell apoptosis rate of 10 μmol/L astaxanthin group was (27.60±4.33)%, which was lower than (38.50±2.38)% of oxidative stress group, but higher than (14.90±1.23)% of 20 μmol/L astaxanthin group and (9.20±0.24)% of normal control group, showing statistically significant differences (all at P<0.05). The NO and MDA contents were higher and the SOD and GSH concentrations were lower in oxidative stress group than in normal control group, 10 μmol/L astaxanthin group and 20 μmol/L astaxanthin group, and the differences were statistically significant (all at P<0.05). The NO and MDA contents were higher and the SOD and GSH concentrations were lower in 10 μmol/L astaxanthin group than in normal control group and 20 μmol/L astaxanthin groups, and the differences were statistically significant (all at P<0.05). There were significant differences in the relative expression levels of nuclear Nrf2, cytoplasmic Nrf2, HO-1 and NQO1 proteins among normal control group, oxidative stress group, 10 μmol/L astaxanthin group and 20 μmol/L astaxanthin group ( F=43.512, 20.381, 31.014, 23.435; all at P<0.001). The relative expression of nuclear Nrf2 protein gradually decreased, and the relative expression of nuclear Nrf2, HO-1 and NQO1 proteins increased gradually in normal control group, oxidative stress group, 10 μmol/L astaxanthin group and 20 μmol/L astaxanthin group, and there were significant differences when compared in pairs (all at P<0.05). The apoptosis rates of Nrf2-siRNA group and Nrf2-siRNA+ astaxanthin group were higher than those of NC-siRNA group and NC-siRNA+ astaxanthin group, and the differences were statistically significant (all at P<0.05). The cell apoptosis rate was higher in NC-siRNA group than in NC-siRNA+ astaxanthin group, showing a statistically significant difference ( P<0.05). There was no significant difference in the apoptosis rate between Nrf2-siRNA+ astaxanthin group and Nrf2-siRNA group ( P>0.05). The NO and MDA concentrations were higher and the SOD and GSH activities were lower in Nrf2-siRNA group than in the NC-siRNA group, with statistically significant differences (all at P<0.05). The NO and MDA concentrations were lower and the SOD and GSH activities were higher in NC-siRNA+ astaxanthin group than in NC-siRNA group and Nrf2-siRNA+ astaxanthin group, and the differences were statistically significant (all at P<0.05). There was no significant difference in NO and MDA concentrations or the SOD and GSH activities between Nrf2-siRNA+ astaxanthin group and Nrf2-siRNA group (all at P>0.05). Conclusions:Astaxanthin enhances the resistance of lens epithelial cells to H 2O 2-induced oxidative stress damage, which may be achieved by activating the Nrf2-related signaling pathway.

Effects of astaxanthin on oxidative stress and inflammatory reaction in rats with traumatic brain injury based on Nrf2/HO-1 signaling pathway / 中国药房

OBJECTIVE To investigate the effects of astaxanthin on oxidative stress and inflammatory reaction in rats with traumatic brain injury （TBI）. METHODS Male SD rats were randomly divided into sham operation group， model group， astaxanthin low-dose group （20 mg/kg）， astaxanthin high-dose group （40 mg/kg）， astaxanthin+ML385 group [astaxanthin 40 mg/kg+ nuclear factor-erythroid 2-related factor 2 （Nrf2） inhibitor ML385 30 mg/kg]， with 14 rats in each group. Except for the sham operation group， TBI model was induced by the modified Feeney free-fall impact method in other groups. The rats in each drug group were given the corresponding drug intragastrically or intraperitoneally， and the rats in the sham operation group and model group were intragastrically given a constant volume of normal saline. The neurological function of rats in each group was scored on the 1st， 3rd and 7th day after drug intervention； on the 7th day of drug intervention， the changes of cerebral histomorphology and inflammatory infiltration score were observed in each group， and the ultrastructure of nerve cells in brain tissue was also observed. The contents of oxidative stress indexes [superoxide dismutase （SOD）， catalase （CAT）， glutathione peroxidase （GSH-Px）， malondialdehyde （MDA）， nitric oxide （NO）] and inflammatory reaction indexes [tumor necrosis factor-α， interleukin-1β （IL-1β）， IL-6， inducible nitric oxide synthase] as well as protein and mRNA expressions of Nrf2， heme oxygenase-1 （HO-1） and NAD（P）H： quinone oxidoreductase 1 （NQO1） were detected in cerebral tissue. RESULTS Compared with the sham operation group， the brain edema of rats in the model group was obvious， accompanied by a large number of inflammatory cells infiltrated， the shape of organelles was damaged and their number was reduced， and the ultrastructure of nerve cells was seriously damaged. The neurological function score， the contents of SOD， CAT， GSH-Px and NO and the relative expression levels of Nrf2， HO-1 and NQO1 protein and mRNA in brain tissue were significantly decreased， while the inflammatory infiltration scores， the contents of MDA and inflammatory reaction indexes were significantly increased （P＜0.05）. Compared with the model group， low-dose and high-dose astaxanthin could significantly improve the pathological status of brain tissue and nerve cells and neurological function scores （except for the first day of drug intervention in the astaxanthin low-dose group）， increase the contents of SOD， CAT， GSH-Px and NO and the relative expression levels of Nrf2， HO-1， NQO1 protein and mRNA in brain tissue in a dose-dependent manner， and reduce inflammatory infiltration scores， the contents of MDA and inflammatory reaction indexes （P＜0.05）. ML385 could significantly inhibit the above effects of astaxanthin （P＜0.05）. CONCLUSIONS Astaxanthin may reduce the oxidative stress of TBI model rats， alleviate the neurological damage and reduce the level of inflammation reaction by activating the Nrf2/HO-1 signaling pathway.

Astaxanthin protects against hearing impairment in diabetic rats

Abstract Objectives: Diabetes Mellitus (DM) causes an increase in oxidative stress that leads to deterioration in auditory functions. Astaxanthine (AST) is known to have strong antioxidant effects. In this study, the aim is to investigate the effect of AST against hearing loss that is due to DM. Methods: This study is an experimental animal study. The study was designed in four groups with 8 animals (n = 8) in each group. The groups were as follows; Control Group (CNT), Diabetic Group (DM), AST applied diabetic group (DM+AST), and AST applied non-diabetic group (AST). Streptozotocin was applied in rats to induce DM. AST was administered by oral gavage. Auditory Brainstem Responses (ABR) and Distortion Product Otoacoustic Emissions (DPOAE) tests were performed on several days of the study. At the end of the study, pro-inflammatory cytokine levels were analyzed in cochlear tissue samples, and Glutathione Peroxidase (GPx), Superoxide Dismutase (SOD), Catalase (CAT) and Malondialdehyde (MDA) levels were measured. Results: When the findings obtained in the ABR and DPOAE tests in the DM group, it was observed that there was a significant deterioration in the hearing sense. This deterioration was not observed in the DM+AST group. In the DM group, GPx, SOD and CAT levels decreased and MDA levels increased in blood and cochlear tissue. Compared to the DM group, it was noted that antioxidant enzyme levels increased and MDA levels decreased in the DM+AST group. Cochlear tissue pro-inflammatory cytokine levels, which increased with DM, were significantly decreased in the DM+AST group. Conclusion: Even though the effects of AST were investigated in a diabetic experimental animal model, if this molecule is proven to be effective in diabetic humans, it can be considered an adjunct therapeutic option with its antioxidant effects. Level of evidence: The level of evidence of this article is 5. This article is an experimental animal and laboratory study.

Hepatopancreatic contributions of lipids and carotenoids to vitellogenesis in the intertidal anomuran crab, Emerita asiatica (Milne Edwards)

In decapod crustaceans, lipids and the associated carotenoid pigments form an integral part of yolk to serve as nutrientsduring embryogenesis. This study reports on the analysis of different lipid classes and the major carotenoids in the ovary,hepatopancreas and hemolymph and their fluctuation during different phases of ovarian maturation in an anomuran crab, Emerita asiatica. Neutral lipids including triglycerides (TG) and free fatty acids (FFA) formed the bulk of ovarian lipids. Important fatty acids are Saturated fatty acids (SFA) 16:0 and 18:0, Monounsaturated fatty acids (MUFA) 16:1n7 and18:1n9, and Polyunsaturated fatty acids (PUFA) 20:5n3 and 22:6n3. While phospholipids increased during maturation, glycolipids decreased. Cholesterol level in ovary increased initially, but declined during later stages. Dominant pigments, ?-carotene and astaxanthin, steadily increased during ovarian maturation within the ovary, although canthaxanthin declineddrastically towards last stage. In hepatopancreas, however, TG and FFA showed gradual decrease during maturation. Palmitic acid, palmitoleic acid and eicosapentaenoic acid are the predominant fatty acids in hepatopancreas, showing asteady decline during ovarian maturation. Other lipid classes such as glycolipids also showed a decline in hepatopancreas. Both ?-carotene and astaxanthin in hepatopancreas declined from the first stage of ovarian development, suggestingtranslocation to ovary. The overall metabolic changes of lipids and carotenoids in hepatopancreas, hemolymph and ovary areindicative of their accumulation within developing eggs to provide metabolic energy and substrates for membrane formation, and to serve as precursors for pigment formation respectively, during embryogenesis.

Astaxanthin attenuates contrast-induced acute kidney injury in rats by inhibiting dynamin-related protein 1-mediated mitochondrial fission through activating SIRT1 pathway / 中华肾脏病杂志

Objective:To investigate whether astaxanthin (AST) down-regulates dynamin-related protein 1 (Drp1) through activating the silent mating type information regulation 2 homolog-1 (SIRT1) signaling pathway, thereby attenuating contrast-induced acute kidney injury.Methods:Forty adult male Sprague-Dawley rats weighing 160-180 g were randomly divided into five groups: sham surgery group (Sham group), contrast medium injury group (CM group), astaxanthin-intervention group (AST+CM group), SIRT1 inhibitor Ex527 intervention group (Ex527+CM group), and astaxanthin combined with Ex527 intervention group (AST+Ex527+CM group). After 72 hours of modeling, heart blood was removed and kidney tissues were collected for follow-up testing. Serum creatinine (Scr), blood urea nitrogen (BUN), and oxidative stress-related indexes total superoxide dismutase (T-SOD) and malondialdehyde (MDA) were measured by biochemistry; hematoxylin and eosin staining was performed to observe the pathological changes in the kidney; mitochondrial morphology and number were observed by transmission electron microscopy; reactive oxygen species (ROS) levels were detected by ROS staining in frozen sections; TUNEL staining was performed to detect apoptosis level. The expression levels of SIRT1, p53, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), Drp1 and apoptosis-related proteins Bcl-2 and Bax were detected by Western blotting.Results:(1) Compared with the CM group, Scr and BUN level were significantly lower, T-SOD level was higher and MDA level was lower in the AST+CM group, while T-SOD level decreased and MDA level increased after the combination of Ex527 (all P<0.05). (2) ROS expression was lower in the AST+CM group compared to the CM group and higher after the combination of Ex527 (both P<0.05). (3) The number of apoptotic cells was significantly reduced in the AST+CM group compared to the CM group and increased after the combination of Ex527 (both P<0.05). (4) The protein expression levels of SIRT1, PGC-1α and Bcl-2 were increased and the protein expression levels of p53, Drp1 and Bax were decreased (all P<0.05) in the AST+CM group compared with the CM group, and the protein expression levels of SIRT1, PGC-1α and Bcl-2 were decreased and the protein expression levels of p53, Drp1 and Bax were increased when Ex527 was combined (all P<0.05). Conclusion:Astaxanthin can inhibit Drp1-mediated mitochondrial fission by activating the SIRT1 pathway, thereby reducing contrast-induced acute kidney injury in rats.

Transcriptome Analysis of Inhibitory Effect of Astaxanthin Against HepG2 Cell Lines / 肿瘤防治研究

Objective To investigate the gene expression differences of hepatocellular carcinoma (HCC) cells treated with astaxanthin and to analyze its biological information. Methods After treated with astaxanthin, the total RNA of HCC cells was extracted with TRIzol reagent. Illumina TruseqTM RNA sample Prep Kit was used for RNA-seq library construction and sequencing. We analyzed the differentially-expressed genes and function enrichments. Results Transcriptomic analysis showed that there were 39 642 566 and 497 155 920 reads in the control group and treatment group, respectively; the proportion of clean reads obtained by filtration were 94.89% and 93.56%, respectively. A total of 77 344 transcripts were detected, with 4 997 genes with significant differences in expression, among which 1 564 genes were up-regulated and 3 433 genes were down-regulated. Conclusions Astaxanthin may participate in several biological processes and signaling pathways of tumors. Significant repression of translation process by astaxanthin may result in the growth inhibition of HCC.

Identification and expression analysis of WRKY gene family in eukaryotic algae / 生物工程学报

WRKY is a superfamily of plant-specific transcription factors, playing a critical regulatory role in multiple biological processes such as plant growth and development, metabolism, and responses to biotic and abiotic stresses. Although WRKY genes have been characterized in a variety of higher plants, little is known about them in eukaryotic algae, which are close to higher plants in evolution. To fully characterize algal WRKY family members, we carried out multiple sequence alignment, phylogenetic analysis, and conserved domain prediction to identify the WRKY genes in the genomes of 30 algal species. A total of 24 WRKY members were identified in Chlorophyta, whereas no WRKY member was detected in Rhodophyta, Glaucophyta, or Bacillariophyta. The 24 WRKY members were classified into Ⅰ, Ⅱa, Ⅱb and R groups, with a conserved heptapeptide domain WRKYGQ(E/A/H/N)K and a zinc finger motif C-X4-5-C-X22-23-H-X-H. Haematococcus pluvialis, a high producer of natural astaxanthin, contained two WRKY members (HaeWRKY-1 and HaeWRKY-2). Furthermore, the coding sequences of HaeWRKY-1 and HaeWRKY-2 genes were cloned and then inserted into prokaryotic expression vector. The recombinant vectors were induced to express in Escherichia coli BL21(DE3) cells and the fusion proteins were purified by Ni-NTA affinity chromatography. HaeWRKY-1 had significantly higher expression level than HaeWRKY-2 in H. pluvialis cultured under normal conditions. High light stress significantly up-regulated the expression of HaeWRKY-1 while down-regulated that of HaeWRKY-2. The promoters of HaeWRKY genes contained multiple cis-elements responsive to light, ethylene, ABA, and stresses. Particularly, the promoter of HaeWRKY-2 contained no W-box specific for WRKY binding. However, the W-box was detected in the promoters of HaeWRKY-1 and the key enzyme genes HaeBKT (β-carotene ketolase) and HaePSY (phytoene synthase) responsible for astaxanthin biosynthesis. Considering these findings and the research progress in the related fields, we hypothesized that the low expression of HaeWRKY-2 under high light stress may lead to the up-regulation of HaeWRKY-1 expression. HaeWRKY-1 may then up-regulate the expression of the key genes (HaeBKT, HaePSY, etc.) for astaxanthin biosynthesis, consequently promoting astaxanthin enrichment in algal cells. The findings provide new insights into further analysis of the regulatory mechanism of astaxanthin biosynthesis and high light stress response of H. pluvialis.

The dietary supplement of Ginkgo biloba: a comprehensive review of its potential interactions based on pre-clinical and clinical evidences / El suplemento dietético de Ginkgo biloba: una revisión exhaustiva de sus posibles interacciones basada en evidencias clínicas y preclínicas

This review present Gingko biloba (GB) interactions, based on clinical and pre-clinical presentations. Literature was retrieved using databases; ScienceDirect, PubMed, Google scholar, Web of Science, Scopus etc. 14/45 interactions were found with clinical presentations. More interactions (80%) were reported with drugs followed by herbs (11.1%), and nutraceuticals (6.7%) with major mechanisms of interaction observed as; inhibition of Cytochrome metabolizing enzymes (44.4%) and platelet-activating factor (PAF) i.e. 15.6%. Major clinical features were; increased bleeding (eye, parietal), hematomas (subdural), and seizures as well as increased blood pressure, priapism, loss of infection/antiviral failure, and coma. Drugs with major interactions belonged to anti-platelet/anti-coagulant and NSAIDs. Synergistic effects were observed for GB vs herbs (except cannabis which showed rhabdomyolysis), foods, and nutraceuticals (except pyridoxine where neurotoxicity was seen). GB use should be monitored and the patient may seek proper advice from a healthcare professional.

Esta revisión presenta las interacciones de Gingko biloba (GB), basadas en presentaciones clínicas y preclínicas. La literatura se recuperó utilizando bases de datos; ScienceDirect, PubMed, Google Scholar, Web of Science, Scopus, etc. Se encontraron 14/45 interacciones con presentaciones clínicas. Se informaron más interacciones (80%) con fármacos seguidos de hierbas (11,1%) y nutracéuticos (6,7%) con los principales mecanismos de interacción observados como; inhibición de las enzimas metabolizadoras del citocromo (44,4%) y factor activador de plaquetas (PAF), es decir, 15,6%. Las principales características clínicas fueron; aumento de sangrado (ojo, parietal), hematomas (subdural) y convulsiones, así como aumento de la presión arterial, priapismo, pérdida de infección / insuficiencia antiviral y coma. Los fármacos con interacciones importantes pertenecían a los antiplaquetarios/anticoagulantes y los AINE. Se observaron efectos sinérgicos para GB frente a hierbas (excepto cannabis que mostró rabdomiólisis), alimentos y nutracéuticos (excepto piridoxina donde se observó neurotoxicidad). Se debe controlar el uso de GB y el paciente puede buscar el asesoramiento adecuado de un profesional de la salud.

Molecular characterization and functional analysis of scavenger receptor class B from black tiger shrimp (Penaeus monodon)

BACKGROUND: Scavenger receptor class B (SRB) is a multifunctional protein in animals that participates in physiological processes, including recognition of a wide range of ligands. Astaxanthin is a major carotenoid found in shrimp. However, the molecular mechanism of astaxanthin and SRB protein binding has not been reported. RESULTS: In the present study, a member of the SRB subfamily, named PmSRB, was identified from the transcriptome of black tiger shrimp (Penaeus monodon). The open reading frame of PmSRB was 1557 bp in length and encoded 518 amino acids. The structure of PmSRB included a putative transmembrane structure at the N-terminal region and a CD36 domain. Multiple sequence alignment indicated that the CD36 domain were conserved. Phylogenetic analysis showed four separate branches (SRA, SRB, SRC, and croquemort) in the phylogenetic tree and that PmSRB was clustered with SRB of Eriocheir sinensis. Quantitative real-time polymerase chain reaction showed that the PmSRB gene was widely expressed in all tissues tested, with the highest expression level observed in the lymphoid organ and brain. Subcellular localization analysis revealed that PmSRB-GFP (green fluorescent protein) fusion proteins were predominantly localized in the cell membrane. The recombinant proteins of PmSRB showed binding activities against astaxanthin in vitro. CONCLUSIONS: PmSRB was identified and characterized in this study. It is firstly reported that PmSRB may take as an important mediator of astaxanthin uptake in shrimp.

Astaxanthin inhibits inflammation of human periodontal ligament cells induced by lipopolysaccharide / 中南大学学报(医学版)

OBJECTIVES@#Human periodontal ligament cells (hPDLCs) are important source of periodontal tissue reconstruction. Under chronic inflammation, the multi-directional differentiation potential and chemotaxis in hPDLCs are decreased. Therefore, inhibiting inflammatory microenvironment and improving the functional characteristics of stem cells can better promote periodontal tissue reconstruction. This study was to investigate the effect of astaxanthin (AST) on lipopolysaccharide (LPS)-induced inflammation in hPDLCs and the underlying mechanisms.@*METHODS@#hPDLCs were isolated and cultured in vitro, and vimentin and keratin immunocytochemical staining were used to identify hPDLCs. CCK-8 assay was used to measure the effects of AST (1, 5, 10, 20, 50, 100, and 200 μmol/L) on proliferation of hPDLCs. Quantitative RT-PCR (RT-qPCR) and ELISA were used to measure the mRNA and protein expression of inflammatory factors (IL-6, IL-1β, and TNF-α) in the control (Con) group, the LPS group, and the LPS+AST (5, 10, 20, and 50 μmol/L) group. Western blotting was used to detect the protein expression of IKBα, phosphorylated IKBα (p-IKBα), and p65 in the Con group, the LPS group, the AST (20 μmol/L) group, and the LPS+AST (20 μmol/L) group. After 10 μmol/L PDTC treatment, the mRNA and protein expressions of IL-6, IL-1β, and TNF-α were detected by RT-qPCR and ELISA.@*RESULTS@#Cell morphology and immunocytochemical staining showed that the cells were in line with the characteristics of hPDLCs. Treatment with AST could promote the proliferation of hPDLCs, which reached the peak at 20 μmol/L. The mRNA and protein expressions of IL-6, IL-1β, and TNF-α in the LPS group were higher than those in the Con group (all @*CONCLUSIONS@#AST promotes the proliferation of hPDLCs, which is related to suppression of LPS-induced the secretion of inflammatory factors via inhibiting the activation of NF-κB signaling pathway.

Transcriptome analysis of signal transduction pathway involved in light inducing astaxanthin accumulation in Haematococcus pluvialis / 生物工程学报

The unicellular green alga Haematococcus pluvialis is the best source of natural astaxanthin (AST) in the world due to its high content under stress conditions. Although high light (HL) can effectively induce AST biosynthesis, the specific mechanisms of light signal perception and transduction are unclear. In the current study, we used transcriptomic data of normal (N), high white light (W), and high blue light (B) to study the mechanisms of light inducing AST accumulation from the point of photoreceptors. The original data of 4.0 G, 3.8 G, and 3.6 G for N, W, and B were obtained, respectively, by the Illumina Hi-seq 2000 sequencing technology. Totally, 51 954 unigenes (at least 200 bp in length) were generated, of which, 20 537 unigenes were annotated into at least one database (NR, NT, KO, SwissProt, Pfam, GO, or KOG). There were 1 255 DEGs in the W vs N, 1 494 DEGs in the B vs N, and 1 008 DEGs in the both W vs N and B vs N. KEGG enrichment analysis revealed that photosynthesis, oxidative phosphorylation, carotenoid biosynthesis, fatty acids biosynthesis, DNA replication, nitrogen metabolism, and carbon metabolism were the significantly enriched pathways. Moreover, a large number of genes encoding photoreceptors and predicted interacting proteins were predicted in Haematococcus transcriptome data. These genes showed significant differences at transcriptional expression levels. In addition, 15 related DEGs were selected and tested by qRT-PCR and the results were significantly correlated with the transcriptome data. The above results indicate that the signal transduction pathway of "light signal - photoreceptors - interaction proteins - (interaction proteins - transcription factor/transcriptional regulator) - gene expression - AST accumulation" might play important roles in the regulation process, and provide reference for further understanding the transcriptional regulation mechanisms of AST accumulation under HL stress.

The SREBP (Sterol Regulatory Element-Binding Protein) pathway: a regulatory bridge between carotenogenesis and sterol biosynthesis in the carotenogenic yeast Xanthophyllomyces dendrorhous

Xanthophyllomyces dendrorhous is a basidiomycete yeast that naturally produces the red-orange carotenoid astaxanthin, which has remarkable antioxidant properties. The biosynthesis of carotenoids and sterols share some common elements that have been studied in X. dendrorhous. For example, their synthesis requires metabolites derived from the mevalonate pathway and in both specific pathways, cytochrome P450 enzymes are involved that share a single cytochrome P450 reductase, CrtR, which is essential for astaxanthin biosynthesis, but is replaceable for ergosterol biosynthesis. Research on the regulation of carotenoid biosynthesis is still limited in X. dendrorhous; however, it is known that the Sterol Regulatory Element-Binding Protein (SREBP) pathway, which is a conserved regulatory pathway involved in the control of lipid metabolism, also regulates carotenoid production in X. dendrorhous. This review addresses the similarities and differences that have been observed between mammal and fungal SREBP pathways and what it is known about this pathway regarding the regulation of the production of carotenoids and sterols in X. dendrorhous.

Liposome Encapsulated Astaxanthin Altered Biochemical Profile In Diethylnitrosamine (DEN) Induced Hepato Carcinoma On Swiss Albino Mice

Cancer is a disease in which a group of abnormal cells grow uncontrollably by disregarding the normal rules of cell division. Across several cancers, Hepatocellular carcinoma (HCC) is one of the most aggressive cancers in worldwide. It is held responsible for up to 1 million deaths globally per annum. HCC is an inflammation-related cancer, as a chronic inflammatory state is necessary for cancer appearance. In this study, the drug astaxanthin and encapsulated astaxanthin was tested against HCC. Mice were divided into 7 groups; Group I: control, Group II: DEN induced, Group III: DEN + 50 mg/kg astaxanthin, Group IV: DEN + 100 mg/kg astaxanthin, Group V: DEN + 50 mg/kg encapsulated astaxanthin, Group VI: DEN + 100 mg/kg encapsulated astaxanthin, Group VII: DEN + 10 mg/kg sorafenib. Regular diet was given. Body weight, Food intake, water intake was noted. Other biochemical parameters such as ALP, AST, Albumin, proteins and TNF-α was determined. Finally, the liver was removed from each mice of different group by sacrificing them and histopathology was done. In vivo evaluation in mice models showed significant antitumor activities by both encapsulated and non-encapsulated astaxanthin at 100 mg/kg as compared with the control, DEN induced group and positive drug sorafenib. This research suggested that encapsulated astaxanthin can also be used as chemotherapeutic agent for the treatment of Hepatocellular carcinoma (HCC).

Engineering CrtW and CrtZ for improving biosynthesis of astaxanthin in Escherichia coli / 中国天然药物

This study engineered β-carotene ketolase CrtW and β-carotene hydroxylase CrtZ to improve biosynthesis of astaxanthin in Escherichia coli. Firstly, crtW was randomly mutated to increase CrtW activities on conversion from β-carotene to astaxanthin. A crtW* mutant with A6T, T105A and L239M mutations has improved 5.35-fold astaxanthin production compared with the wild-type control. Secondly, the expression levels of crtW* and crtZ on chromosomal were balanced by simultaneous modulation RBS regions of their genes using RBS library. The strain RBS54 selected from RBS library, directed the pathway exclusively towards the desired product astaxanthin as predominant carotenoid (99%). Lastly, the number of chromosomal copies of the balanced crtW-crtZ cassette from RBS54 was increased using a Cre-loxP based technique, and a strain with 30 copies of the crtW*-crtZ cassette was selected. This final strain DL-A008 had a 9.8-fold increase of astaxanthin production compared with the wild-type control. Fed-batch fermentation showed that DL-A008 produced astaxanthin as predominant carotenoid (99%) with a specific titer of 0.88 g·L without addition of inducer. In conclusion, through constructing crtW mutation, balancing the expression levels between crtW* and crtZ, and increasing the copy number of the balanced crtW*-crtZ cassette, the activities of β-carotene ketolase and β-carotene hydroxylase were improved for conversion of β-carotene to astaxanthin with higher efficiency. The series of conventional and novel metabolic engineering strategies were designed and applied to construct the astaxanthin hetero-producer strain of E. coli, possibly offering a general approach for the construction of stable hetero-producer strains for other natural products.

Extraction and purification of astaxanthin from shrimp shells and the effects of different treatments on its content

Abstract In this study, the extraction process and purification technology of astaxanthin from shrimp shells were established, and the effects of different treatments on the content of astaxanthin were studied. The determination results of astaxanthin in various shrimp/crab shells showed that the astaxanthin content in the Procambarus clarkia shell reached 239.96 µg/g. The effects of cool-ventilated, sun-dried and cooking conditions on the content of astaxanthin during the treatment of shrimp shells were investigated respectively and fresh shrimp shells as best extraction source was determined. The nine groups orthogonal test design with four factors and three levels (L9(3)4) was used to analyze the optimization of extraction process of astaxanthin from shrimp shells with ethanol as an environmentally friendly extraction solvent. The optimum experimental condition including the solid-liquid ratio (1:7), extraction time (20 min) and temperature (50 ºC) was proposed with the maximum extraction yield of astaxanthin. Next, silica gel column chromatography was used to purify the crude extraction of astaxanthin, and the purity of astaxanthin increased from 0.34% to 85.1% (about 250 times), which has great applications in the high value utilization of shrimp shells resources and the development of astaxanthin-related products.

Astaxanthin Prevents Decreases in Superoxide Dismutase 2 Level and Superoxide Dismutase Activity in Helicobacter pylori-infected Gastric Epithelial Cells

BACKGROUND: Helicobacter pylori increases production of reactive oxygen species (ROS), which activates inflammatory and carcinogenesis-related signaling pathways in gastric epithelial cells. Therefore, reducing ROS, by upregulating antioxidant enzyme, such as superoxide dismutase (SOD), may be a novel strategy to prevent H. pylori-associated gastric diseases. Astaxanthin is an antioxidant carotenoid that prevents oxidative stress-induced cell injury. The present study was aimed to determine whether H. pylori decreases SOD activity by changing the levels of SOD1/SOD2 and whether astaxanthin prevents changes in SOD levels and activity in H. pylori-infected gastric epithelial AGS cells. METHODS: AGS cells were pre-treated with astaxanthin for 3 hours prior to H. pylori infection and cultured for 1 hour in the presence of H. pylori. SOD levels and activity were assessed by Western blot analysis and a commercial assay kit, respectively. Mitochondrial ROS was determined using MitoSOX fluorescence. RESULTS: H. pylori decreased SOD activity and the SOD2 level, but increased mitochondrial ROS in AGS cells. The SOD1 level was not changed by H. pylori infection. Astaxanthin prevented H. pylori-induced decreases in the SOD2 level and SOD activity and reduced mitochondrial ROS in AGS cells. CONCLUSIONS: Consumption of astaxanthin-rich food may prevent the development of H. pylori-associated gastric disorders by suppressing mitochondrial oxidative stress.

Combination Effect of Titrated Extract of Centella asiatica and Astaxanthin in a Mouse Model of Phthalic Anhydride-Induced Atopic Dermatitis

PURPOSE: In our previous study, we demonstrated that both titrated extract of Centella asiatica (TECA) and astaxanthin (AST) have anti-inflammatory effects in a 5% phthalic anhydride (PA) mouse model of atopic dermatitis (AD). The increasing prevalence of AD demands new therapeutic approaches for treating the disease. We investigated the therapeutic efficacy of the ointment form of TECA, AST and a TECA + AST combination in a mouse model of AD to see whether a combination of the reduced doses of 2 compounds could have a synergistic effect. METHODS: An AD-like lesion was induced by the topical application of 5% PA to the dorsal ear and back skin of an Hos:HR-1 mouse. After AD induction, TECA (0.5%), AST (0.5%) and the TECA (0.25%) + AST (0.25%) combination ointment (20 μg/cm2) were spread on the dorsum of the ear or back skin 3 times a week for 4 weeks. We evaluated dermatitis severity, histopathological changes and changes in protein expression by Western blotting for inducible nitric oxide synthase (iNOS), cyclocxygenase (COX)-2, and nuclear factor (NF)-κB activity. We also measured the concentrations of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and immunoglobulin E (IgE) in the blood of AD mice by enzyme-linked immunosorbent assay (ELISA). RESULTS: PA-induced skin morphological changes and ear thickness were significantly reduced by TECA, AST and TECA + AST treatments, but these inhibiting effects were more pronounced in the TECA + AST treatment. TECA, AST and the TECA+AST reatments inhibited the expression of iNOS and COX-2; NF-κB activity; and the release of TNF-α, IL-6 and IgE. However, the TECA+AST treatment showed additive or synergistic effects on AD. CONCLUSIONS: Our results demonstrate that the combination of TECA and AST could be a promising therapeutic agent for AD by inhibiting NF-κB signaling.

Advances in astaxanthin biosynthesis in Haematococcus pluvialis / 生物工程学报

Astaxanthin is widely applied as a nutraceutical, pharmaceutical, and aquaculture feed additive because of its high antioxidant activity. Haematococcus pluvialis is a microalgal species that can largely accumulate astaxanthin under adverse environmental conditions. Here we review the research progress of astaxanthin biosynthesis in H. pluvialis, including the induction and regulation of massive astaxanthin, the relationship between astaxanthin synthesis, photosynthesis and lipid metabolism.

Effects of astaxanthin on the expression of NF-κB inflammatory signaling pathway related proteins in hippocampus of rats after acute carbon monoxide poisoning / 中华行为医学与脑科学杂志

Objective@#To investigate the effects of astaxanthin on neuronal injury in hippocampus of rats after acute carbon monoxide poisoning(ACMP) and the relationship with NF-κB inflammatory signaling pathway.@*Methods@#Male SD rats screened by water maze were randomly divided into three group(n=50): control group (NC group), CO poisoning group (COP group), CO poisoning+ astaxanthin group (AST group) . ACMP rat model was established by static inhaled exposure method. Meanwhile, rats in AST group were further given astaxanthin twice a day by gavage.At 1 day, 7 days, 14 days, 21 days and 28 days after CO poisoning (10 rats in each group were selected), the learning and memory abilities were evaluated by Morris water maze test. The pathological changes of the neurons in hippocampal CA1 region were observed by hematoxylin-eosin(HE) staining.The expression and activation of NF-κB in hippocampus were detected by immunoblotting and immunofluorescence, and the expression of TNF-α and IL-6 protein in hippocampus were examined by ELISA.@*Results@#Morris water maze test showed that there were no significant difference in escape latency and crossing platform times between the three groups (P>0.05). Compared with NC group, the escape latency of COP group was prolonged at 14, 21 and 28 days after CO poisoning (t=-6.04, -6.28, -8.18, all P<0.05), and the number of crossing platform was decreased (t= 5.96, 7.85, 6.51, all P<0.05). Compared with the COP group, the escape latency of the rats in AST group at the 14, 21 and 28 days was shortened (t=4.74, 4.82, 5.98, all P<0.05), and the number of crossing platform was increased (t=-3.72, -4.45, -6.53, all P<0.05). Compared with NC group, the number of NF-κ B positive cells in CA1 area of hippocampus in COP group increased at every time point (t=-8.62, -18.00, -16.67, -11.15, -6.22, all P<0.05); the number of NF-κ B positive cells in CA1 area in AST group decreased at 7 d, 14 d, 21 d and 28 d after CO poisoning, the difference was statistically significant (t= 6.55, 6.96, 4.40, 4.17; all P<0.05). Western blot showed that the changes of NF-κB protein was similar to that of immunofluorescence. After 7 days of CO poisoning, the level of NF-κB protein in hippocampus of COP group was (1.44±0.08), it was higher than that of NC group (t=-20.07, P<0.05), while that of AST group was (0.68±0.10), it was lower than that of COP group (t=10.23, P<0.05). The results of Elisa showed that TNF-α and IL-6 in the hippocampus of COP group were higher than those of NC group at every time point(all P<0.05), while compared with COP group, TNF-α and IL-6 in AST group were lower (all P<0.05). After 7 days of CO poisoning, TNF -α in COP group ((39.04±5.29) pg/ml)was higher than that in NC group ((14.13±2.12) pg/ml) (t=-7.58, P<0.05); TNF -α in AST group ((25.77±3.31) pg/ml) was lower than that in COP group (t=3.69, P<0.05). After 7 days of CO poisoning, the level of IL-6 in COP group ((181.79±9.12) pg/ml)was higher than that in NC group ((73.12±11.04) pg/ml) (t=-8.24, P<0.05), and the level of IL-6 in AST group ((121.47±9.80) pg/ml) was lower than that in COP group (t=7.80, P<0.05).@*Conclusion@#The excessive inflammatory response which mediated by NF-κB signaling pathway is involved in ACMP-induced neuronal damage in hippocampus.Astaxanthin can down-regulate the expression of NF-κB inflammatory signaling pathway related proteins, and pave a way for the treatment of ACMP brain damage and cognitive dysfunction.

Proliferation and invasion effect of astaxanthin on human ovarian cancer and its mechanism / 国际生物医学工程杂志

Objective To investigate the effects of astaxanthin on the proliferation and invasion of SKOV3 human ovarian cancer cells and to explore its mechanism. Methods MTT colorimetry was used to investigate the inhibition effect of astaxanthin on SKOV3 cells. Transwell assay was used to investigate the effect of astaxanthin on the invasion of SKOV3 cells. Flow cytometry was used to investigate the cell cycle of astaxanthin on SKOV3 cells. The effect of astaxanthin on cell cycle and invasion-related protein expression was investigated by Western Blot method. Results Astaxanthin had a significant inhibitory effect on cell proliferation and was concentration dependent. The invasive ability of SKOV3 cells was significantly decreased under the treatment of astaxanthin(P<0.05). Compared with control group, the proportion of SKOV3 cells in G1 phase in the astaxanthin-treated group was significantly increased. The protein expression of matrix metallo proteinase 2(MMP-2), matrix metallo proteinase 9 (MMP-9), and cyclin-dependent protein kinase 6(CDK6) and Cyclin A were significantly decreased in astaxanthin-treated SKOV3 cells compared with the control group ( all P<0 . 05 ) . Conclusions Astaxanthin can significantly inhibit the growth of SKOV3 cells and arrest the SKOV3 cell cycle in G1 phase, and can inhibit the growth and invasion of SKOV3 by regulating cell cycle and invasion-related proteins.