Caffeic acid dimethyl ether alleviates alcohol-induced hepatic steatosis via microRNA-378b-mediated CaMKK2-AMPK pathway.

Alcoholic liver disease (ALD), with its increasing morbidity and mortality, has seriously and extensively affected the health of people worldwide. Caffeic Acid Dimethyl Ether (CADE) significantly inhibits alcohol-induced hepatic steatosis in vivo through AMP-activated protein kinase (AMPK) pathway, but its in-depth mechanism remains unclear. This work aimed to clarify further mechanism of CADE in improving hepatic lipid accumulation in ALD through the microRNA-378b (miR-378b)-mediated Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2)-AMPK signaling pathway. Here, we reported that the hepatic or serum triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), and aspartate transaminase (AST) levels were sharply escalated by ethanol while prominently decreased by CADE. Ethanol sharply up-regulated miR-378b expression while CADE effectively prevented the elevation of miR-378b in vivo. And treatment of CADE surely increased mRNA and protein expression of CaMKK2 as a kinase of AMPK and reduced lipid accumulation in the livers of alcohol-fed C57BL/6 mice. MiR-378b escalation exacerbated hepatic steatosis and inhibited CaMKK2-AMPK signaling, while miR-378b deficiency alleviated lipid accumulation and activated the CaMKK2 cascade. Furthermore, CADE alleviated the lipid deposition and reversed the disorder of CaMKK2-AMPK signaling pathway induced by miR-378b over-expression. However, knockdown of miR-378b eliminated the beneficial effect of CADE on lipid metabolism. In brief, our results showed that CADE ultimately improved hepatic lipid deposition by regulating the CaMKK2-AMPK signaling pathway through miR-378b.

Methyl ferulic acid ameliorates alcohol-induced hepatic insulin resistance via miR-378b-mediated activation of PI3K-AKT pathway.

A previous study indicated that microRNA-378b (miR-378b) plays a critical role in controlling hepatic insulin resistance by targeting insulin receptor (IR) and p110α in alcoholic liver disease (ALD). Methyl ferulic acid (MFA), a bioactive ingredient in Securidaca inappendiculata Hassk rhizomes, exhibits multiple pharmacological activities. It has been reported that MFA ameliorates insulin resistance in ALD, whereas the underlying molecular mechanism remains unclear. The objective of study was to evaluate the influence of MFA on insulin sensitivity in ethanol-induced L-02 cells as well as alcohol-fed mice and illuminate the function of miR-378b-mediated phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) pathway in system. MFA was found to remarkably down-regulate miR-378b level and increase IR and p110α expressions. Furthermore, the effect of MFA on modulating miR-378b/PI3K-AKT pathway to enhance insulin sensitivity was corroborated by overexpressing and inhibiting miR-378b. Taken together, MFA exhibited a positive effect against ALD by attenuating the inhibition of miR-378b on IR/p110α and partly activating the insulin signaling to alleviate alcohol-induced hepatic insulin resistance.

microRNA-378b regulates ethanol-induced hepatic steatosis by targeting CaMKK2 to mediate lipid metabolism.

Alcoholic liver disease (ALD) has seriously harmed the health of people worldwide, but its underlying mechanisms remain unclear. This study aims to clarify the biological function of microRNA-378b (miR-378b) in ethanol (EtOH)-induced hepatic lipid accumulation. Here, we report miR-378b is over-expressed in EtOH-induced cells and EtOH-fed mice and finally accelerates lipid accumulation. MiR-378b directly targets Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2), a kinase of AMP-activated protein kinase (AMPK), and mediates the protein level of CaMKK2. Over-expression of miR-378b exacerbated the lipid accumulation induced by EtOH and inhibited CaMKK2 and the AMPK cascade while inhibition of miR-378b ameliorated lipid metabolism dysfunction in vivo and in vitro. In brief, our results show that miR-378b plays an important role in the regulation of lipid metabolism by directly targeting CaMKK2.

Characterization of Starch in Cucurbita moschata Germplasms throughout Fruit Development.

Pumpkins (Cucurbita moschata; Cucurbitaceae) are the rich source of nutrients and valued for their biologically active substances to be used for the treatment of several diseases. The contents, composition, and conformation of starch are the significant quality traits of C. moschata. Two germplasms were targeted for analysis regarding the taste difference. Results indicated that the total starch contents and amylose/amylopectin ratio were high in CMO-X as compared to CMO-E during each fruit development stage. Scanning electron microscopy and transmission electron microscopy observations revealed that smooth surface starch granules fused together to enhance the starch accumulation. For a comparison of fruit development in CMO-E and CMO-X, the putative pathway for starch metabolism was developed and homologs were identified for each key gene involved in the pathway. GBSS and SBE were correlated with the difference in the amylose/amylopectin ratio of CMO-E and CMO-X. Conclusively, the developmental regulation of genes associated with starch accumulation can be considered as an important factor for the determination of fruit quality.

miR-378b Regulates Insulin Sensitivity by Targeting Insulin Receptor and p110α in Alcohol-Induced Hepatic Steatosis.

Insulin resistance has been implicated in alcoholic liver disease. A previous study has shown that microRNAs (miRNAs) play a major role in the production, secretion, and function of insulin. MiRNAs are capable of repressing multiple target genes that in turn negatively regulate various physiological and pathological activities. However, current information on the biological function of miRNAs in insulin resistance is limited. The goal of the present study was to elucidate the role of miR-378b in alcohol-induced hepatic insulin resistance and its underlying mechanism. This study has observed that miR-378b is up-regulated in National Institute on Alcohol Abuse and Alcoholism (NIAAA) alcoholic mouse models as well as in ethanol-induced L-02 cells in vitro. Furthermore, miR-378b overexpression impaired the insulin signaling pathway, and inhibition of miR-378b improved insulin sensitivity in vivo and in vitro. A mechanistic study revealed that IR and p110α are direct targets of miR-378b. Together, these results suggest that miR-378b controls insulin sensitivity by targeting the insulin receptor (IR) as well as p110α and possibly play an inhibitory role in the development of insulin resistance, thereby providing insights into the development of novel diagnostic and treatment methods.

Metabolic and transcriptomic analysis of two Cucurbita moschata germplasms throughout fruit development.

BACKGROUND: Pumpkins (Cucurbita moschata; Cucurbitaceae) are valued for their fruits and seeds and are rich in nutrients. Carotenoids and sugar contents, as main feature of pumpkin pulp, are used to determine the fruit quality. RESULTS: Two pumpkin germplasms, CMO-X and CMO-E, were analyzed regarding the essential quality traits such as dry weight, soluble solids, organic acids, carotenoids and sugar contents. For the comparison of fruit development in these two germplasms, fruit transcriptome was analyzed at 5 different developmental stages from 0 d to 40 d in a time course manner. Putative pathways for carotenoids biosynthesis and sucrose metabolism were developed in C. moschata fruit and homologs were identified for each key gene involved in the pathways. Gene expression data was found consistent with the accumulation of metabolites across developmental stages and also between two germplasms. PSY, PDS, ZEP, CRTISO and SUS, SPS, HK, FK were found highly correlated with the accumulation of carotenoids and sucrose metabolites, respectively, at different growth stages of C. moschata as shown by whole transcriptomic analysis. The results of qRT-PCR analysis further confirmed the association of these genes. CONCLUSION: Developmental regulation of the genes associated with the metabolite accumulation can be considered as an important factor for the determination of C. moschata fruit quality. This research will facilitate the investigation of metabolic profiles in other cultivars.

Ethanol-Induced Hepatic Insulin Resistance is Ameliorated by Methyl Ferulic Acid Through the PI3K/AKT Signaling Pathway.

One of the key events during the development of alcoholic liver disease (ALD) is that alcohol inhibits the insulin signaling pathway in liver and leads to disorders of glucose and lipid metabolism. Methyl ferulic acid (MFA) is a biologically active monomer isolated from the root of Securidaca inappendiculata Hasskarl. It has been reported that MFA has a hepatoprotective effect against alcohol-induced liver injury in vivo and in vitro. However, the effect of MFA on ethanol-induced insulin resistance in ALD remains unclear. In this study, we investigated whether MFA could exert protective effects against hepatic insulin resistance in ethanol-induced L-02 cells and ALD rats. ALD was induced in vivo by feeding Lieber-DeCarli diet containing 5% (w/v) alcohol for 16 weeks to Sprague-Dawley rats. Insulin resistance was induced in vitro in human hepatocyte L-02 cells with 200 mM ethanol for 24 h followed by 10-7 nM insulin for 30 min. MFA exhibited the effects of inhibited insulin resistance, reduced enzymatic capacity for hepatic gluconeogenesis, and increased hepatic glycogen synthesis both in vivo and in vitro. In addition, the results of transcriptome sequencing of liver tissues in the ethanol- and MFA-treated groups indicated that "pyruvate metabolism," "glycolysis/gluconeogenesis," and "fatty acid metabolism" were significantly different between ethanol- and MFA-treated groups. Further studies suggested that MFA activated the hepatic phosphatidylinositol 3-kinase (PI3K)/AKT pathway in vivo and in vitro. Taken together, these findings suggested that MFA effectively ameliorated hepatic insulin resistance in ALD at least partially by acting on the PI3K/AKT pathway.

A high-density genetic map developed by specific-locus amplified fragment (SLAF) sequencing and identification of a locus controlling anthocyanin pigmentation in stalk of Zicaitai (Brassica rapa L. ssp. chinensis var. purpurea).

BACKGROUND: Caixin and Zicaitai (Brassica rapa) belong to Southern and Central China respectively. Zicaitai contains high amount of anthocyanin in leaf and stalk resulting to the purple color. Stalk is the major edible part and stalk color is an economically important trait for the two vegetables. The aim of this study is to construct a high density genetic map using the specific length amplified fragment sequencing (SLAF-seq) technique to explore genetic basis for anthocyanin pigmentation traits via quantitative trait loci (QTL) mapping. RESULTS: We constructed a high generation linkage map with a mapping panel of F2 populations derived from 150 individuals of parental lines "Xianghongtai 01" and "Yinong 50D" with purple and green stalk respectively. The map was constructed containing 4253 loci, representing 10,940 single nucleotide polymorphism (SNP) markers spanning 1030.04 centiMorgans (cM) over 10 linkage groups (LGs), with an average distance between markers of 0.27 cM. Quantitative trait loci (QTL) analysis revealed that a major locus on chromosome 7 and 4 minor QTLs explaining 2.69-61.21% of phenotypic variation (PVE) were strongly responsible for variation in stalk color trait. Bioinformatics analysis of the major locus identified 62 protein-coding genes. Among the major locus, there were no biosynthetic genes related to anthocyanin. However, there were several transcription factors like helix-loop-helix (bHLH) bHLH, MYB in the locus. Seven predicted candidate genes were selected for the transcription level analysis. Only bHLH49 transcription factor, was significantly higher expressed in both stalks and young leaves of Xianghongtai01 than Yinong50D. An insertion and deletion (InDel) marker developed from deletion/insertion in the promoter region of bHLH49 showed significant correlation with the stalk color trait in the F2 population. CONCLUSION: Using the constructed high-qualified linkage map, this study successfully identified QTLs for stalk color trait. The identified valuable markers and candidate genes for anthocyanin accumulation in stalk will provide useful information for molecular regulation of anthocyanin biosynthesis. Overall our findings will lay a foundation for functional gene cloning, marker-assisted selection (MAS) and molecular breeding of important economic traits in B. rapa.

Methyl ferulic acid attenuates liver fibrosis and hepatic stellate cell activation through the TGF-ß1/Smad and NOX4/ROS pathways.

Liver fibrosis is a pathological wound-healing response caused by chronic liver damage due to a virus, autoimmune disorder, or drugs. Hepatic stellate cells (HSCs) play an essential role in the pathogenesis of liver fibrosis. Methyl ferulic acid (MFA), a biologically active monomer, has a protective effect on liver injury. However, the effects and roles of MFA in liver fibrosis remain unknown. The purpose of the current study was to investigate the effect of MFA on hepatic fibrosis and the underlying mechanisms. Human hepatic stellate LX-2 cells were exposed to 5 µg/L TGF-ß1 for 48 h to stimulate liver fibrosis in vitro. Using MTT, RT-PCR and Western blot analysis, we revealed that MFA significantly inhibited the proliferation of LX-2 cells as well as decreased the expressions of α-SMA and type I collagen in LX-2 cells. SD rats were fed with ethanol, and this combined with the intraperitoneal injection of CCl4 induced liver fibrosis in vivo. We found that the administration of MFA markedly decreased the levels of hyaluronic acid (HA), procollagen type III (PC-III), type IV collagen (CIV) and laminin (LN) in the serum, inhibited the expression of α-smooth muscle actin (α-SMA) as well as type I and type III collagen, and up-regulated the ratio of MMP-2/TIMP-1 in rats. The antifibrotic effects of MFA were also evaluated by H&E staining and Masson's trichrome staining. In addition, further studies suggested that this protection by MFA from liver fibrosis was possibly related to the inhibition of TGF-ß1/Smad and NOX4/ROS signalling. In conclusion, our results demonstrate that MFA attenuated liver fibrosis and hepatic stellate cell activation by inhibiting the TGF-ß1/Smad and NOX4/ROS signalling pathways.

Methyl ferulic acid attenuates ethanol-induced hepatic steatosis by regulating AMPK and FoxO1 Pathways in Rats and L-02 cells.

Methyl ferulic acid (MFA) is a biologically active monomer extracted and purified from the Chinese herbal medicine Securidaca inappendiculata hasskarl. The previously studies showed that MFA improved acute liver injury induced by ethanol. However, the effect of MFA on ethanol-induced hepatic steatosis in alcoholic liver disease (ALD) still remains unclear. The current study was aimed at elucidating the effect of MFA on alcohol-induced hepatic steatosis and the underlying mechanisms. Human hepatocyte L-02 cells exposed to 200 mM ethanol for 24 h to simulate alcoholic steatosis in vitro. SD rats were fed a Lieber-DeCarli diet containing 5% (w/v) alcohol for 16 weeks to induce alcoholic liver disease in vivo. We examined the effect of MFA on ethanol-induced lipid deposition in L-02 cells and SD rats. The results showed that MFA reduced the accumulation of lipid in L-02 cells, improved alcoholic liver injury in rats, alleviated hepatic pathological lesions, and reduced lipid deposition in rat serum and liver. Further studies suggest that MFA reduces lipid synthesis by activating AMPK-ACC/MAPK-FoxO1 pathway. In addition, MFA also promotes lipid oxidation by up-regulating the expression of SIRT1, PPAR-α, and CPT-1α. Taken together, MFA ameliorates ethanol-induced hepatic steatosis by activating AMPK-ACC/MAPK-FoxO1 pathway and up-regulating the expression levels of SIRT1, PPAR-α, and CPT-1α.

NOX4/ROS mediate ethanol­induced apoptosis via MAPK signal pathway in L­02 cells.

The aim of the present study was to assess the molecular mechanism of ethanol­induced oxidative stress­mediated apoptosis in L­02 liver cells in order to elucidate novel pathways associated with alcoholic liver disease. L­02 cells were treated with 400 mM ethanol with or without inhibitors. The cell viability was measured by an MTT assay. Cell apoptosis was assessed by flow cytometry and a single­stranded DNA (ssDNA) assay. Intracellular reactive oxygen species (ROS) production of L­02 cells was determined using the 2',7'­dichlorofluorescein­diacetate dye. The protein expression of c­Jun N­terminal kinase (JNK), phosphorylated (p)­JNK, P38, p­P38, NADPH oxidase (NOX)1, NOX4, p22phox, B­cell lymphoma 2 (Bcl­2) and Bcl­2­associated X protein were measured by western blot analysis. The mRNA expression of NOX1, NOX4 and p22phox was measured by reverse transcription polymerase chain reaction analysis. The results indicated that after treatment with various concentrations of ethanol for the indicated durations, L­02 cells were displayed a significant decrease in cell viability in a dose­and time­dependent manner. Ethanol­induced apoptosis and cell death of L­02 cells was accompanied by the generation of ROS, elevated expression of NOX, as well as phosphorylation of JNK and P­38. In addition, increased expression of Bcl­2 was induced by 400 mM ethanol. Furthermore, treatment with NOX inhibitor attenuated the ethanol­induced a decrease in cell viability, and an increase in apoptosis and Bcl­2 expression. In conclusion, ethanol induced apoptosis in the L­02 hepatocyte cell line via generation of ROS and elevated expression of NOX4. This indicated that activation of JNK and p38 in the mitogen­activated protein kinase pathway promotes apoptosis in L­02 cells.

A high-density linkage map and QTL mapping of fruit-related traits in pumpkin (Cucurbita moschata Duch.).

Pumpkin (Cucurbita moschata) is an economically worldwide crop. Few quantitative trait loci (QTLs) were reported previously due to the lack of genomic and genetic resources. In this study, a high-density linkage map of C. moschata was structured by double-digest restriction site-associated DNA sequencing, using 200 F2 individuals of CMO-1 × CMO-97. By filtering 74,899 SNPs, a total of 3,470 high quality SNP markers were assigned to the map spanning a total genetic distance of 3087.03 cM on 20 linkage groups (LGs) with an average genetic distance of 0.89 cM. Based on this map, both pericarp color and strip were fined mapped to a novel single locus on LG8 in the same region of 0.31 cM with phenotypic variance explained (PVE) of 93.6% and 90.2%, respectively. QTL analysis was also performed on carotenoids, sugars, tuberculate fruit, fruit diameter, thickness and chamber width with a total of 12 traits. 29 QTLs distributed in 9 LGs were detected with PVE from 9.6% to 28.6%. It was the first high-density linkage SNP map for C. moschata which was proved to be a valuable tool for gene or QTL mapping. This information will serve as significant basis for map-based gene cloning, draft genome assembling and molecular breeding.

Hepatoprotective effects of Methyl ferulic acid on alcohol-induced liver oxidative injury in mice by inhibiting the NOX4/ROS-MAPK pathway.

AIMS: The present study aimed to investigate the hepatoprotective effects of Methyl ferulic acid (MFA) against oxidative stress and apoptosis as well as inflammation in mice with liver injury induced by alcohol and its underlying mechanisms. METHODS: C57BL/6 mice were divided into a control group,a model group, and Methyl ferulic acid with high dosage (20 mg/kg), moderate dosage (10 mg/kg) and low dosage (5 mg/kg) groups. The general condition and organ index of each group were investigated. Histopathological analysis was performed to determine the degree of hepatic injury. Biochemical analyses of functional liver enzymes, lipid peroxidation enzymes and lipid content in each group. The levels of inflammatory cytokines were measured by enzyme-linked immunosorbent assay (ELISA). The mechanisms were investigated by detecting levels of NADPH Oxidase 4 (NOX4),p22phox, cytochrome P4502E1 (CYP2E1),Bax,B-cell lymphoma 2 (Bcl-2),cleaved-caspase 3 and 9 and phosphorylated extracellular regulated protein kinases(ERK),phosphorylated c-Jun N-terminal kinase (JNK), and phosphorylated p38 mitogen-activated protein kinase (MAPK) using real-time polymerase chain reaction (PCR) and Western blotting. RESULTS: MFA treatment significantly decreased serum enzymatic activities of alanine aminotransferase (ALT) and aspartate aminotransaminase (AST). MFA markedly increased levels of superoxide dismutase (SOD), catalase (CAT), glutathione (GSH), glutathione peroxidase (GSH-Px) and total antioxidative capacity (T-AOC), and reduced the concentration of malondialdehyde (MDA) and reactive oxygen species (ROS). Histopathological examination of livers showed that MFA reduced cytoplasmic vacuolisation necrosis and inflammatory cell infiltration in alcohol-treated mice. MFA treatment remarkably reduced the levels of trigyceride (TG), total cholesterol (TC) and low-density lipoprotein (LDL), decreasing the levels of high-density lipoprotein (HDL), alcohol dehydrogenase(ADL) and aldehyde dehydrogenase (ALDH). MFA treatment remarkably inhibited the expression of inflammatory factors tumour necrosis factor (TNF)-α, monocyte chemoattractant protein 1 (MCP-1), interleukin (IL)-1ß and IL-6. MFA attenuated both mRNA and protein expression of NOX4,p22phox,CYP2E1,Bax/Bcl-2. In addition, MFA inhibited the activation of caspase 3 and 9 and downregulated the levels of p-JNK,p-p38 MAPK and p-ERK in liver. CONCLUSION: MFA has a protective effect on alcohol-induced liver injury, which may be related to its antioxidant,anti-inflammatory,lipid-eliminating properties and its ability to regulate the NOX4/ROS-MAPK signalling pathway.

Metabolic engineering of tomato for high-yield production of astaxanthin.

Dietary carotenoids have been shown to be beneficial to health by decreasing the risk of many diseases. Attempts to enhance carotenoids in food crops have been successful although higher plants appear to resist big changes of carotenoid biosynthesis by metabolic engineering. Here we report the generation of a more nutritious tomato by modifying the intrinsic carotenes to astaxanthin, a high-value ketocarotenoid rarely found in plants. This was achieved by co-expression of the algal ß-carotene ketolase from Chlamydomonas reinhardtii and ß-carotene hydroxylase from Haematococcus pluvialis, a unique pair of enzymes identified to co-operate perfectly in converting ß-carotene to astaxanthin by functional complementation in Escherichia coli. Expression of the two enzymes in tomato up-regulated most intrinsic carotenogenic genes, and efficiently directed carbon flux into carotenoids, leading to massive accumulations of mostly free astaxanthin in leaves (3.12mg/g) but esterified astaxanthin in fruits (16.1mg/g) and a 16-fold increase of total carotenoid capacity therein without affecting the plant normal growth and development. This study opened up the possibility of employing crop plants as green factories for economical production of astaxanthin.

Functional characterization of various algal carotenoid ketolases reveals that ketolating zeaxanthin efficiently is essential for high production of astaxanthin in transgenic Arabidopsis.

Extending the carotenoid pathway to astaxanthin in plants is of scientific and industrial interest. However, expression of a microbial ß-carotene ketolase (BKT) that catalyses the formation of ketocarotenoids in transgenic plants typically results in low levels of astaxanthin. The low efficiency of BKTs in ketolating zeaxanthin to astaxanthin is proposed to be the major limitation for astaxanthin accumulation in engineered plants. To verify this hypothesis, several algal BKTs were functionally characterized using an Escherichia coli system and three BKTs were identified, with high (up to 85%), moderate (∼38%), and low (∼1%) conversion rate from zeaxanthin to astaxanthin from Chlamydomonas reinhardtii (CrBKT), Chlorella zofingiensis (CzBKT), and Haematococcus pluvialis (HpBKT3), respectively. Transgenic Arabidopsis thaliana expressing the CrBKT developed orange leaves which accumulated astaxanthin up to 2 mg g(-1) dry weight with a 1.8-fold increase in total carotenoids. In contrast, the expression of CzBKT resulted in much lower astaxanthin content (0.24 mg g(-1) dry weight), whereas HpBKT3 was unable to mediate synthesis of astaxanthin in A. thaliana. The none-native astaxanthin was found mostly in a free form integrated into the light-harvesting complexes of photosystem II in young leaves but in esterified forms in senescent leaves. The alteration of carotenoids did not affect chlorophyll content, plant growth, or development significantly. The astaxanthin-producing plants were more tolerant to high light as shown by reduced lipid peroxidation. This study advances a decisive step towards the utilization of plants for the production of high-value astaxanthin.