Integration of Transcriptome and Metabolome Provides New Insights to Flavonoids Biosynthesis in Dendrobium huoshanense.

Dendrobium huoshanense is both a traditional herbal medicine and a plant of high ornamental and medicinal value. We used transcriptomics and metabolomics to investigate the effects of growth year on the secondary metabolites of D. huoshanense stems obtained from four different years of cultivation. In this study, a total of 428 differentially accumulated metabolites (DAMs) and 1802 differentially expressed genes (DEGs) were identified. The KEGG enrichment analysis of DEGs and DAMs revealed significant differences in "Flavonoid biosynthesis", "Phenylpropanoid biosynthesis" and "Flavone and flavonol biosynthesis". We summarize the biosynthesis pathway of flavonoids in D. huoshanense, providing new insights into the biosynthesis and regulation mechanisms of flavonoids in D. huoshanense. Additionally, we identified two candidate genes, FLS (LOC110107557) and F3'H (LOC110095936), which are highly involved in flavonoid biosynthesis pathway, by WGCNA analysis. The aim of this study is to investigate the effects of growth year on secondarily metabolites in the plant and provide a theoretical basis for determining a reasonable harvesting period for D. huoshanense.

Transcriptome and metabolome profiling unveil the accumulation of flavonoids in Dendrobium officinale.

Dendrobium officinale is a Chinese herbal medicine with a long history of use in China. Flavonoids are known to be an important secondary metabolite in Dendrobium officinale, but very little is known about their molecular regulation mechanism in D. officinale. In this study, we collected one to four years old D. officinale stems for the purpose of RNA-sequencing and mass spectrometry data collection. The results showed that metabolome analysis detected 124 different flavonoid metabolites of which flavonol metabolites were significantly increased in biennial samples. In the transcriptome analysis, 30 different genes involved in the synthesis of flavonoid were identified. The key genes FLS (LOC110101392, LOC110107557, LOC110114894) that regulate the synthesis of flavonols are highly expressed in biennial samples. The present study contributes a new insight into the molecular mechanism of flavonoid accumulation in D. officinale.

Procyanidin B2 Reduces Vascular Calcification through Inactivation of ERK1/2-RUNX2 Pathway.

Vascular calcification is strongly associated with atherosclerotic plaque burden and plaque instability. The activation of extracellular signal-regulated kinase 1/2 (ERK1/2) increases runt related transcription factor 2 (RUNX2) expression to promote vascular calcification. Procyanidin B2 (PB2), a potent antioxidant, can inhibit ERK1/2 activation in human aortic smooth muscle cells (HASMCs). However, the effects and involved mechanisms of PB2 on atherosclerotic calcification remain unknown. In current study, we fed apoE-deficient (apoE-/-) mice a high-fat diet (HFD) while treating the animals with PB2 for 18 weeks. At the end of the study, we collected blood and aorta samples to determine atherosclerosis and vascular calcification. We found PB2 treatment decreased lesions in en face aorta, thoracic, and abdominal aortas by 21.4, 24.6, and 33.5%, respectively, and reduced sinus lesions in the aortic root by 17.1%. PB2 also increased α-smooth muscle actin expression and collagen content in lesion areas. In the aortic root, PB2 reduced atherosclerotic calcification areas by 75.8%. In vitro, PB2 inhibited inorganic phosphate-induced osteogenesis in HASMCs and aortic rings. Mechanistically, the expression of bone morphogenetic protein 2 and RUNX2 were markedly downregulated by PB2 treatment. Additionally, PB2 inhibited ERK1/2 phosphorylation in the aortic root plaques of apoE-/- mice and calcified HASMCs. Reciprocally, the activation of ERK1/2 phosphorylation by C2-MEK1-mut or epidermal growth factor can partially restore the PB2-inhibited RUNX2 expression or HASMC calcification. In conclusion, our study demonstrates that PB2 inhibits vascular calcification through the inactivation of the ERK1/2-RUNX2 pathway. Our study also suggests that PB2 can be a potential option for vascular calcification treatment.

Apigenin protects mice against 3,5-diethoxycarbonyl-1,4-dihydrocollidine-induced cholestasis.

Cholestasis can induce liver fibrosis and cirrhosis. Apigenin has anti-oxidant and anti-inflammatory effects. Herein, we determined whether apigenin can protect mice against cholestasis. In vitro, apigenin protected TFK-1 cells (a human bile duct cancer cell line) against H2O2-induced ROS generation and inhibited transforming growth factor-ß-activated collagen type 1 alpha 1 and α-smooth muscle actin in LX2 cells (a human hepatic stellate cell line). In vivo, cholestatic mice induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) were treated with apigenin. Apigenin potently blocked DDC-induced gallbladder atrophy and associated liver injury, fibrosis and collagen accumulation. Moreover, apigenin relieved the DDC-caused abnormality of bile acid metabolism and restored the balance between bile secretion and excretion by regulating the farnesoid X receptor signaling pathway. Furthermore, apigenin reduced inflammation or oxidative stress in the liver by blocking the DDC-activated Toll-like receptor 4, nuclear factor κB and tumor necrosis factor α, or DDC-suppressed superoxidase dismutase 1/2, catalase and glutathione peroxidase. Taken together, apigenin improves DDC-induced cholestasis by reducing inflammation and oxidative damage and improving bile acid metabolism, indicating its potential application for cholestasis treatment.

NaoXinTong Capsule ameliorates memory deficit in APP/PS1 mice by regulating inflammatory cytokines.

Alzheimer's disease (AD) is the most common neurodegenerative disease in aging population. Neuroinflammation, hyperphosphorylated Tau (p-Tau) and the imbalance between production and clearance of ß-amyloid peptide (Aß) are the major causes for AD development. NaoXinTong Capsule (NXT), a traditional Chinese medicine, is wildly used for treatment of cardiovascular and cerebrovascular diseases. Hence, we used the double transgenic mice expressing chimeric human amyloid precursor protein and mutant human presenilin 1 (APP/PS1) and HT-22 cells to determine the neuroprotective effects of NXT in AD development and the involved mechanisms. The 3-month-old APP/PS1 mice were randomly divided into 3 groups and received following treatment: Control group, mice were fed normal chow; NXT groups, mice were fed normal chow containing NXT at a normal and a high dose, respectively. While the age-matched C57BL/6J mice fed normal chow were used as the normal control. The NXT treatment was lasted for 5 months. We found that NXT treatment improved spatial memory impairment and cognitive decline in APP/PS1 mice by decreasing p-Tau levels and Aß accumulation in the brain. Mechanistically, we observed that NXT inhibited neuron atrophy and apoptosis by downregulating inflammatory cytokines, interleukin 1ß (IL-1ß), IL-6 and tumor necrosis factor α (TNF-α), and inflammation mediators, nuclear factor κB (NF-κB) and toll-like receptor 4 (TLR4) in the brain. Consistently, NXT blocked l-glutamic acid-induced reactive oxygen species production, inflammation and apoptosis in HT-22 cells partially by inhibiting TLR4/NF-κB/IL-1ß signaling pathway. Our study demonstrates that NXT ameliorates AD by reducing p-Tau, Aß accumulation, inflammation and neuron apoptosis via regulation of TLR4-mediated inflammatory system. It also suggests the potential application of NXT for AD treatment.

LongShengZhi Capsule Attenuates Alzheimer-Like Pathology in APP/PS1 Double Transgenic Mice by Reducing Neuronal Oxidative Stress and Inflammation.

Alzheimer's disease (AD) is the most common form of dementia in the elderly. It may be caused by oxidative stress, inflammation, and cerebrovascular dysfunctions in the brain. LongShengZhi Capsule (LSZ), a traditional Chinese medicine, has been approved by the China Food and Drug Administration for treatment of patients with cardiovascular/cerebrovascular disease. LSZ contains several neuroprotective ingredients, including Hirudo, Astmgali Radix, Carthami Flos (Honghua), Persicae Semen (Taoren), Acori Tatarinowii Rhizoma (Shichangpu), and Acanthopanax Senticosus (Ciwujia). In this study, we aimed to determine the effect of LSZ on the AD process. Double transgenic mice expressing the amyloid-ß precursor protein and mutant human presenilin 1 (APP/PS1) to model AD were treated with LSZ for 7 months starting at 2 months of age. LSZ significantly improved the cognition of the mice without adverse effects, indicating its high degree of safety and efficacy after a long-term treatment. LSZ reduced AD biomarker Aß plaque accumulation by inhibiting ß-secretase and γ-secretase gene expression. LSZ also reduced p-Tau expression, cell death, and inflammation in the brain. Consistently, in vitro, LSZ ethanol extract enhanced neuronal viability by reducing L-glutamic acid-induced oxidative stress and inflammation in HT-22 cells. LSZ exerted antioxidative effects by enhancing superoxide dismutase and glutathione peroxidase expression, reduced Aß accumulation by inhibiting ß-secretase and γ-secretase mRNA expression, and decreased p-Tau level by inhibiting NF-κB-mediated inflammation. It also demonstrated neuroprotective effects by regulating the Fas cell surface death receptor/B-cell lymphoma 2/p53 pathway. Taken together, our study demonstrates the antioxidative stress, anti-inflammatory, and neuroprotective effects of LSZ in the AD-like pathological process and suggests it could be a potential medicine for AD treatment.

Ascorbic acid enhances low-density lipoprotein receptor expression by suppressing proprotein convertase subtilisin/kexin 9 expression.

Ascorbic acid, a water-soluble antioxidant, regulates various biological processes and is thought to influence cholesterol. However, little is known about the mechanisms underpinning ascorbic acid-mediated cholesterol metabolism. Here, we determined if ascorbic acid can regulate expression of proprotein convertase subtilisin/kexin 9 (PCSK9), which binds low-density lipoprotein receptor (LDLR) leading to its intracellular degradation, to influence low-density lipoprotein (LDL) metabolism. At cellular levels, ascorbic acid inhibited PCSK9 expression in HepG2 and Huh7 cell lines. Consequently, LDLR expression and cellular LDL uptake were enhanced. Similar effects of ascorbic acid on PCSK9 and LDLR expression were observed in mouse primary hepatocytes. Mechanistically, ascorbic acid suppressed PCSK9 expression in a forkhead box O3-dependent manner. In addition, ascorbic acid increased LDLR transcription by regulating sterol regulatory element-binding protein 2. In vivo, administration of ascorbic acid reduced serum PCSK9 levels and enhanced liver LDLR expression in C57BL/6J mice. Reciprocally, lack of ascorbic acid supplementation in L-gulono-γ-lactone oxidase deficient (Gulo-/-) mice increased circulating PCSK9 and LDL levels, and decreased liver LDLR expression, whereas ascorbic acid supplementation decreased PCSK9 and increased LDLR expression, ameliorating LDL levels in Gulo-/- mice fed a high fat diet. Moreover, ascorbic acid levels were negatively correlated to PCSK9, total and LDL levels in human serum samples. Taken together, these findings suggest that ascorbic acid reduces PCSK9 expression, leading to increased LDLR expression and cellular LDL uptake. Thus, supplementation of ascorbic acid may ameliorate lipid profiles in ascorbic acid-deficient species.

LongShengZhi capsule inhibits doxorubicin-induced heart failure by anti-oxidative stress.

Heart failure is a major cause of morbidity and mortality worldwide. LongShengZhi capsule (LSZ), a traditional Chinese medicine, is used for treatment of patients with vascular diseases. Herein we investigated the effect of LSZ treatment on doxorubicin (DOX)-induced heart failure in mice. C57BL/6 mice randomly in 3 groups received following treatment: Control group, mice were fed normal chow; DOX group, mice were intraperitoneally injected DOX to induce heart failure and fed normal chow; and LSZ group, mice were injected DOX and fed normal chow containing LSZ. DOX induced heart failure as evidenced by increased serum creatine kinase, lactic dehydrogenase and α-hydroxybutyrate dehydrogenase, and cardiac fibrosis. However, LSZ treatment substantially inhibited DOX-induced heart failure parameters. Mechanistically, LSZ reduced collagen content and fibrosis by inhibiting expression of collagen type I α1 (COL1α1), COL1α2, α-smooth muscle actin and transforming growth factor ß1. In addition, DOX-induced cell apoptosis was inhibited by LSZ, coupled with reduced caspase 3 activity and mRNA expression. LSZ decreased inflammatory cytokine levels. More importantly, LSZ decreased oxidative stress by inducing expression of anti-oxidative stress enzymes including superoxide dismutase 1 (SOD1), SOD2, catalase and glutathione peroxidase 1 through activation of forkhead box O3A and sirtuin 3. In conclusion, our study demonstrates that LSZ reduces heart failure by reducing production of reactive oxygen species and inhibiting inflammation/apoptosis. Our study also suggests the potential application of LSZ for heart failure treatment.

Comparative nutritional characteristics of the three major Chinese Dendrobium species with different growth years.

Dendrobium, an important medicinal plant, is a source of widely used herbal medicine to nourish the stomach and treat throat inflammation. The present study is aimed at distinguishing and evaluating three major Dendrobium species by comparing physiochemical characteristics and understanding differences between different growth years in the Ta-pieh Mountains. Polysaccharides and total alkaloids of Dendrobium were determined, and the amino acids and trace elements were determined by UPLC (Ultra High-Performance Liquid Chromatography) and ICP-MS (Inductively coupled plasma mass spectrometry). It can be seen from the results that the polysaccharide content of these three kinds of Dendrobium in different growth years ranges from 249.31 mg·g-1 to 547.66 mg·g-1, and the highest content is in the 3-year-old Dendrobium huoshanense. The total alkaloid content ranges from 0.21 mg·g-1 to 0.54 mg·g-1, and the highest content is also the 3-year-old Dendrobium huoshanense. We determined the amino acid content of these three Dendrobium in different growth years, and we can see that each of the three kinds of Dendrobium contain seven kinds of amino acids required by the human body. We conducted a safety evaluation of the essential trace elements of Dendrobium, and the results showed that the dosage of 12g·d-1 Dendrobium prescribed in China Pharmacopoeia is in accordance with the recommended daily intake of trace elements recommended by the Food and Drug Administration of the United States, and will not cause trace element poisoning. Linear discriminant analysis was carried out on the basis of amino acids and trace elements and confirmed the applicability of multi-elemental analysis for identifying different Dendrobium species.

Analysis of Dendrobium huoshanense transcriptome unveils putative genes associated with active ingredients synthesis.

BACKGROUND: Dendrobium huoshanense C.Z. Tang et S.J. Cheng is a traditional Chinese herbal medicine with high medicinal value in China. Polysaccharides and alkaloids are its main active ingredients. To understand the difference of main active ingredients in different tissues, we determined the contents of polysaccharides and alkaloids in the roots, stems and leaves of D. huoshanense. In order to explore the reasons for the differences of active ingredients at the level of transcription, we selected roots, stems and leaves of D. huoshanenese for transcriptome sequencing and pathway mining. RESULTS: The contents of polysaccharides and alkaloids of D. huoshanense were determined and it was found that there were significant differences in different tissues. A total of 716,634,006 clean reads were obtained and 478,361 unigenes were assembled by the Illumina platform sequencing. We identified 1407 carbohydrate-active related unigenes against CAZy database including 447 glycosyltransferase genes (GTs), 818 glycoside hydrolases (GHs), 60 carbohydrate esterases (CEs), 62 carbohydrate-binding modules (CBMs), and 20 polysaccharide lyases (PLs). In the glycosyltransferases (GTs) family, 315 differential expression genes (DEGs) were identified. In total, 124 and 58 DEGs were associated with the biosynthesis of alkaloids in Dh_L vs. Dh_S and Dh_R vs. Dh_L, respectively. A total of 62 DEGs associated with the terpenoid pathway were identified between Dh_R and Dh_S. Five key enzyme genes involved in the terpenoids pathway were identified, and their expression patterns in different tissues was validated using quantitative real-time PCR. CONCLUSIONS: In summary, our study presents a transcriptome profile of D. huoshanense. These data contribute to our deeper relevant researches on active ingredients and provide useful insights into the molecular mechanisms regulating polysaccharides and alkaloids in Dendrobium.