Erucic acid from Isatis indigotica Fort. suppresses influenza A virus replication and inflammation in vitro and in vivo through modulation of NF-kB and p38 MAPK pathway

Isatis indigotica Fort.(Ban-Lan-Gen)is an herbal medicine prescribed for influenza treatment.However,its active components and mode of action remain mostly unknown.In the present study,erucic acid was isolated from Isatis indigotica Fort.,and subsequently its underlying mechanism against influenza A virus(IAV)infection was investigated in vitro and in vivo.Our results demonstrated that erucic acid exhibited broad-spectrum antiviral activity against IAV resulting from reduction of viral polymerase transcription activity.Erucic acid was found to exert inhibitory effects on IAV or viral(v)RNA-induced pro-inflam-matory mediators as well as interferons(IFNs).The molecular mechanism by which erucic acid with antiviral and anti-inflammatory properties was attributed to inactivation of NF-kB and p38 MAPK signaling.Furthermore,the NF-kB and p38 MAPK inhibitory effect of erucic acid led to diminishing the transcriptional activity of interferon-stimulated gene factor 3(ISGF-3),and thereby reducing IAV-triggered pro-inflammatory response amplification in IFN-β-sensitized cells.Additionally,IAV-or vRNA-triggered apoptosis of alveolar epithelial A549 cells was prevented by erucic acid.In vivo,erucic acid administration consistently displayed decreased lung viral load and viral antigens expression.Meanwhile,erucic acid markedly reduced CD8+cytotoxic T lymphocyte(CTL)recruitment,pro-apoptotic signaling,hyperactivity of multiple signaling pathways,and exacerbated immune inflammation in the lung,which resulted in decreased lung injury and mortality in mice with a mouse-adapted A/FM/1/47-MA(H1N1)strain infection.Our findings provided a mechanistic basis for the action of erucic acid against IAV-mediated inflammation and injury,suggesting that erucic acid may have a therapeutic potential in the treatment of influenza.

Molecular evidence for blocking erucic acid synthesis in rapeseed(Brassica napus L.) by a two-base-pair deletion in FAE1(fatty acid elongase 1)

DNA sequences of fatty acid elongase 1 genes FAE1.1 （EA） and FAE1.2 （Ec） were isolated and characterized for 30 com- mercialized low erucic acid rapeseed （LEAR） cultivars in China. Four types of independent mutation leading to low erucic acid trait were found, i.e., a single-base transition （eAl）, a two-base deletion （ec2） and four-base deletion （eC4） as well as single-base transition with a four-base deletion （eA.）. Three genotypes, i.e., eA1eA1eC2eC2, eA1eA1eC4eC4 and eA.eA.ec4ec4 were responsible for LEA content in storage Iipids of different rapeseed cultivars. Most of the LEAR cultivars had a genotype of eA1eA1ec2ec2, which were descended from the first LEAR cultivar, Oro. Yeast expression analysis revealed that two-base-pair （AA） deletion （ec2） at the base sites of 1 422-1 423 in the C genome FAE1 gene resulted in the absence of the condensing enzyme and led to the failure to produce erucic acid. Coexpression of FAE1 and ketoacyI-CoA reductase （KCR） or enoyI-CoA reductase （ECR） was found in high erucic acid rapeseed （HEAR） but not in LEAR （eA1eA1ec2ec2oreA1eA1ec4ec4）. Moreover, KCR and ECR were still coordinately regulated in eA1eA1ec2ec2 or eA1eA1ec4ec4 genotypes, suggesting that the expression of two genes was tightly linked. In addition, specific detection methods were developed by high-resolution melting curve analysis in order to detect eA1 and ec4.

Inheritance of Erucic Acid,Glucosinolate,and Oleic Acid Contents in Rapeseed (Brassica napus L.)

This study was conducted to verify the inheritance of certain characters of rapeseed including erucic acid, glucosinolate and oleic acid contents by using generation mean analysis. The cross of lines Ⅲ174×Zi20 （F1）, F2, BC1 （F1×P1）, BC2 （F1×P2）, and parents （P1 and P2） were evaluated in the field. Data were measured on individual plants for oleic acid, erucic acid, and glucosinolate contents. Transgressive variations in F2 population were observed for oleic acid content, indicating that dominance and recessive genes distributed in both parents. Scaling test indicated that the effects of genes controlling these characters did not follow the additivedominance model. The data for three characters were analyzed using six parameter models and found that one or more types of epistatic gene effects were important for glucosinolate content. High broad sense heritabilities were obtained for erucic acid, oleic acid, and glucosinolate contents with the values of 98.97%, 93.68%, and 86.17%, respectively. Two major gene pairs were found to control the expression of erucic acid and oleic acid contents, while three major gene pairs were detected to control glucosinolate content.

Variation and Correlation of Erucic Acid,Oleic Acid and Glucosinolate Contents in Brassica rapa Seeds

In order to screen and identify excellent breeding resources and provide basic materials for Brassica rapa breeding,the contents of erucic acid,oleic acid and glucosinolate in 84 kinds of B. rapa were determined by near-infrared spectroscopy,and the correlations among them were also analyzed. The results showed that the content of erucic acid and oleic acid were significantly negatively correlated,the contents of erucic acid and glucosinolate were significantly positively correlated,while the contents of oleic acid and glucosinolate were significantly negatively correlated; principal component analysis( PCA) were performed on the population materials,factors 1 and 2 were extracted for plotting,factor 1 and factor 2 could explain 73. 7% and 23. 2% of the phenotypic variation,respectively; through cluster analysis,79 materials aggregated to form group I,and five special variants deviated from the population. The variation of erucic acid,oleic acid and glucosinolates in B. rapa populations was rich and there was significant correlation. Through cluster analysis,five excellent B. rapa breeding materials( No. 32,No. 45,No. 46,No. 50,and No. 59) were screened.

Biochemical and histopathological profiling of Wistar rat treated with Brassica napus as a supplementary feed

Metabolic changes together with cardiovascular and hepatic factors are related to the development of diseases like myocardial lipidosis,heart disease,and profound toxicity.The aim of this animal study is to determine the effects of high erucic acid containing rapeseed oil(Brassica napus L.)varieties on liver,kidney and heart muscles in Wistar rats.Male Wistar rats were divided into three groups where each group containing four rats.Group A was considered as control diet group,while Group B rapeseed wild oil group and Group C rapeseed hybrid oil group were considered as experimental diet groups.The levels of aspartate aminotransferase(AST),alanine aminotransferase(ALT),alkaline phosphatase(ALP),creatine kinase-MB(CK-MB)and creatinine of two experimental groups were significantly elevated while compared to the control groups(p<0.05–0.001).Nevertheless,an increment in weight retardation(p<0.05)was also observed in rapeseed hybrid oil treated groups.No significant weight retardation found in other two groups(p>0.05).Noticeable tissue injury observed in this study is a sign of the relative toxicity of erucic acid containing rapeseed oil to mammalian species.The use of Brassica napus as a supplementary feed ingredient should be,therefore,thoroughly considered.

Diversity and association analysis of important agricultural trait based on miniature inverted-repeat transposable element specific marker in Brassica napus L.

Miniature inverted-repeat transposable elements(MITEs)are a group of DNA transposable element(TE)which preferentially distributed with gene associated regions.Tens of MITEs families have been revealed in Brassica napus genome,they scatter across the genome with tens of thousands copies and produce polymorphisms both intra-and inter-species.Our previous studies revealed a Tourist-like MITE,Monkey King,associated with vernalization requirement of B.napus,however there are still few studies reveal MITE association with agricultural traits in B.napus.In the present study,80 polymorphic markers were developed from 55 MITEs,and used to evaluate genetic diversity in a panel of B.napus accessions consisting of 101 natural and 25 synthetic genotypes.Five agricultural traits,oil content,glucosinolate content,erucic acid content,weight of thousand seeds(WTS)and plant height,were investigated across 3-years field experiments,in addition,two traits,hypocotyl length and root length,were evaluated at the 4-leaf stage in the laboratory.Correlations between the MITE-based markers and seven traits were analyzed,finally,10 polymorphic markers produced by 6 pairs of MITE specific primers were revealed relatively high correlation with 5 traits.Two polymorphic markers were anchored with two candidate genes,BnaA02g13530D and BnaA08g20010D,respectively,which may contribute to glucosinolate content and WTS.This research may contribute to genetic improvement through utilization of MITE-induced polymorphisms in Brassica species.

Intergeneric Somatic Hybridization Between Brassica napus L. and Sinapis alba L.

Electrically induced protoplast fusion was used to produce somatic hybrids between Brassica napus L. and Sinapis alba L. Seven hybrids were obtained and verified by the simple sequence repeat and cleaved amplified polymorphic sequence analysis of the gene fael, indicating that the characteristic bands from S. alba were present in the hybrids. The hybridity was also confirmed by chromosome number counting because the hybrids possessed 62 chromosomes, corresponding to the sum of fusion-parent chromosomes. Chromosome pairing at meiosis was predominantly normal, which led to high pollen fertility, ranging from 66% to 77%. All hybrids were grown to full maturity and could be fertilized and set seed after self-pollination or back-crosses with B. napus. The morphology of the hybrids resembled characteristics from both parental species. An analysis of the fatty acid composition in the seeds of F1 plants was conducted and the seeds were found to contain different amounts of erucic acid, ranging from 11.0% to 52.1%.