Impacts of Angelica Polysaccharide on Proliferation and Differentiation of Mesenchymal Stem Cells of Rat Bone Marrow

In literature, antiosteoporotic effects of Angelica sinensis root have been confirmed, but the impact of Angelica sinensis polysaccharide (ASP) on osteoblastic or adipogenic distinction of BMSCs is limited. This paper aimed to explore the role of ASP on proliferation and differentiation of rat BMSCs. Rat BMSCs were subjected to isolation and identification through flow cytometry. The proliferation of rat BMSCs under ASP was performed by CCK-8 kit. Measures of osteogenesis under different concentrations of ASP were detected by using alizarin red staining for mesenchymal cells differentiation and ALP activity assay to identify ALP activity. Quantitative RT-PCR was selected to identify osteoblastic or adipogenic biomarkers from a genetic perspective. Likewise, we have evaluated measures of indicators of Wnt/β-catenin signal. ASP significantly promoted the proliferation, increased osteogenesis, and decreased adipogenesis of rat BMSCs within the limit of 20–60 mg/L in a dose-dependent manner but was suppressed at 80 mg/L. The expression of cyclin D1 and ß-catenin showed a considerable rise over the course of ASP induced osteogenesis. Dickkopf 1 (DKK1) suppressed the regulation of rat BMSCs differentiation through the mediation of ASP. We have observed that ASP upregulated the osteogenic but downregulated adipogenic differentiation of BMSCs, and our findings help to contribute to effective solutions for treating bone disorders.

Cool Temperature Enhances Growth, Ferulic Acid and Flavonoid Biosynthesis While Inhibiting Polysaccharide Biosynthesis in Angelica sinensis

Angelica sinensis, a perennial herb that produces ferulic acid and phthalides for the treatment of cardio-cerebrovascular diseases, prefers growing at an altitude of 1800–3000 m. Geographical models have predicted that high altitude, cool temperature and sunshade play determining roles in geo-authentic formation. Although the roles of altitude and light in yield and quality have been investigated, the role of temperature in regulating growth, metabolites biosynthesis and gene expression is still unclear. In this study, growth characteristics, metabolites contents and related genes expression were investigated by exposing A. sinensis to cooler (15 °C) and normal temperatures (22 °C). The results showed that plant biomass, the contents of ferulic acid and flavonoids and the expression levels of genes related to the biosynthesis of ferulic acid (PAL1, 4CLL4, 4CLL9, C3H, HCT, CCOAMT and CCR) and flavonoids (CHS and CHI) were enhanced at 15 °C compared to 22 °C. The contents of ligustilide and volatile oils exhibited slight increases, while polysaccharide contents decreased in response to cooler temperature. Based on gene expression levels, ferulic acid biosynthesis probably depends on the CCOAMT pathway and not the COMT pathway. It can be concluded that cool temperature enhances plant growth, ferulic acid and flavonoid accumulation but inhibits polysaccharide biosynthesis in A. sinensis. These findings authenticate that cool temperature plays a determining role in the formation of geo-authentic and also provide a strong foundation for regulating metabolites production of A. sinensis.

Integrated Metabolomic and Transcriptomic Analysis Reveals Differential Mechanism of Flavonoid Biosynthesis in Two Cultivars of Angelica sinensis

Angelica sinensis is a traditional Chinese medicinal plant that has been primarily used as a blood tonic. It largely relies on its bioactive metabolites, which include ferulic acid, volatile oils, polysaccharides and flavonoids. In order to improve the yield and quality of A. sinensis, the two cultivars Mingui 1 (M1), with a purple stem, and Mingui 2 (M2), with a green stem, have been selected in the field. Although a higher root yield and ferulic acid content in M1 than M2 has been observed, the differences of flavonoid biosynthesis and stem-color formation are still limited. In this study, the contents of flavonoids and anthocyanins were determined by spectrophotometer, the differences of flavonoids and transcripts in M1 and M2 were conducted by metabolomic and transcriptomic analysis, and the expression level of candidate genes was validated by qRT-PCR. The results showed that the contents of flavonoids and anthocyanins were 1.5- and 2.6-fold greater in M1 than M2, respectively. A total of 26 differentially accumulated flavonoids (DAFs) with 19 up-regulated (UR) and seven down-regulated (DR) were obtained from the 131 identified flavonoids (e.g., flavonols, flavonoid, isoflavones, and anthocyanins) in M1 vs. M2. A total 2210 differentially expressed genes (DEGs) were obtained from the 34,528 full-length isoforms in M1 vs. M2, and 29 DEGs with 24 UR and 5 DR were identified to be involved in flavonoid biosynthesis, with 25 genes (e.g., CHS1, CHI3, F3H, DFR, ANS, CYPs and UGTs) mapped on the flavonoid biosynthetic pathway and four genes (e.g., RL1, RL6, MYB90 and MYB114) belonging to transcription factors. The differential accumulation level of flavonoids is coherent with the expression level of candidate genes. Finally, the network of DAFs regulated by DEGs was proposed. These findings will provide references for flavonoid production and cultivars selection of A. sinensis.

Physiological and Biochemical Responses, and Comparative Transcriptome Profiling of Two Angelica sinensis Cultivars Under Enhanced Ultraviolet-B Radiation

In this study, we explored the adaptive mechanism of two varieties of Angelica sinensis exposed to enhanced Ultraviolet-B (UV-B) radiation. The radiation had different effects on the biomass, photosynthetic performance, oxidative damage, antioxidant defense system, and levels of bioactive compounds of Mingui 1 (C1) and Mingui 2 (C2). C2 outperformed C1 under enhanced UV-B radiation, compared to natural light. Using the Illumina RNA-seq, we obtained 6,326 and 2,583 DEGs in C1 and C2, respectively. Under enhanced UV-B radiation, the mRNA levels of genes involved in photosynthesis, antennae protein synthesis, carbon fixation, chlorophyll synthesis, and carotenoid synthesis were decreased in C1 but stable in C2, involving few DEGs. TFs were widely involved in the response of C1 to enhanced UV-B radiation; almost all bHLH and MYB coding genes were downregulated whereas almost all genes encoded WRKY22, WRKY50, WRKY72, NCF, and HSF were upregulated. These results indicate that enhanced UV-B radiation was not conducive to the synthesis of flavonoids, while disease resistance was enhanced. Regarding the ROS scavenging system, upregulated DEGs were mainly found in the AsA-GSH cycle and PrxR/Trx pathways. Remarkably, DEGs that those encoding biosynthetic key enzymes, including ferulic acid (CHS, CHI, DFR, and ANS) and flavonoid (CHS, CHI, DFR, and ANS), most upregulation in C2, leading to increased accumulation of ferulic acid and flavonoids and adversely affecting C1. Genes encoding key enzymes involved in the synthesis of lactone components (ACX, PXG) were mostly up-regulated in C1, increasing the content of lactone components. Our results reveal the DEGs present between C1 and C2 under enhanced UV-B radiation and are consistent with the observed differences in physiological and biochemical indexes. C1 was more sensitive to enhanced UV-B radiation, and C2 was more tolerant to it under moderate enhanced UV-B radiation stress. In addition, the large amount of A. sinensis transcriptome data generated here will serve as a source for finding effective ways to mitigate UV-B enhancement, and also contribute to the well-established lack of genetic information for non-model plant species.

Angelica sinensis polysaccharide promotes the proliferation and osteogenic differentiation of human dental pulp stem cells (hDPSCs) by activating the wnt/β-catenin pathway

Human dental pulp stem cells (hDPSCs) are capable of forming mineralized nodules. The proliferation and osteogenic differentiation of hDPSCs are very important for alleviating tooth defects caused by related diseases. Angel-ica polysaccharide (ASP) is the main bioactive ingredient extracted from the angelica root. ASP has a variety of biological functions, including immune regulation, antitumor activity, and hematopoiesis. However, its possible effects on hDPSCs are still unclear. In this study, we aimed to investigate the role of ASP in periodontal diseases. We found that ASP promoted the proliferation of hDPSCs and osteogenic differentiation of hDPSCs. We further found that it promoted the expression of osteogenic-related genes, including ALP, RUNX2, Col1a1, and OCN. Mechanically, we found that ASP activated the Wnt/β-catenin pathway. In conclusion, our results suggested that ASP promoted the proliferation and osteogenic differentiation of hDPSCs via the Wnt/β-catenin pathway.

First report of Fusarium avenaceum causing Leaf Spot on Angelica sinensis in China

Angelica sinensis (Oliv) Diels (Umbelliferae) is a popular Chinese herb that is mainly distributed in Gansu Province, China, accounting for more than 90% of the national output and sales. A survey for diseases of A. sinensis in Gansu Province in August 2019 found foliar disease with an incidence of 60 to100%, and severities ranging from 5 to 15%. The disease mainly occurred in late July and August. The initial symptoms included many light brown, small lesions, round or irregular in shape, which gradually increased in size. White mycelia was visible in the lesions. Severely affected leaves became chlorotic, withered and died. In the Angelica planting area in Weiyuan County (33°26′N, 104°02′E) diseased leaves from 20 plants were collected by the five-point sampling method (Zheng et al. 2018), and small samples (4 × 4 mm2) wee cut from the border between diseased and healthy tissue, successively sterilized with 75% ethanol for 30 sec, washed three times with sterilized water and dried on sterilized filter paper, and placed on potato dextrose agar plates. After 5 days at 25°C, five morphologically similar colonies were obtained. Colonies were somewhat round with pink overall and formed abundant fluffy white mycelium in the center. Conidia were solitary, macrospores slender, straight to slightly falcate with 2 to 6 septa, and ranged from 20.0 to 77.6 µm × 2.5 to 3.6 µm (n=50). The microspores were elliptical and ranged from 3.0 to 8.0 µm × 2.5 to 3.0 µm (n=5). The strong pink pigment was observed on the reverse side of the PDA culture. The morphological characteristics were consistent with the description of Fusarium avenaceum (Parikh et al. 2018; Jahedi et al. 2019). To further identify the strains, the internal transcribed spacer (ITS), β-tubulin, translation elongation factor 1α (EF1-α), and RNA polymerase second largest subunit (RPB2) gene regions were amplified with ITS1/ITS4, Bt2a/Bt2b, EF1/EF2, and 5f2/7cr (Glass and Donaldson 1995; O’Donnell et al. 2010; White et al. 1990), respectively. The sequences of the five strains were identical, and that of representative strain K0721 were deposited in GenBank (ITS, MZ389899; TUB2, MZ398139; EF1-α, MZ388462; RPB2, MZ394004). BLAST analysis revealed that the ITS, β-tubulin, EF1-α, and RPB2 sequences were 100% (563/563), 100% (423/423), 99% (643/649), and 99% (930/935) homology, with those of F. avenaceum (KP295511.1, KY475586.1, KU999088.1, and MH582082.1), respectively. A multigene phylogenetic tree was inferred by Maximum likelihood phylogenetic analyses based on the combined data set with ITS, EF1-α and RPB2. The strain K0721 was clustered with F. avenaceum. Pathogenicity tests were performed on five 1-month-old healthy plants in plastic pots (20 cm. diam.) with sterilized soil. Each was sprayed with 50 μl of a conidial suspension (1×104 conidia/mL), and 5 healthy plants were sprayed with sterile water as controls. Small lesions were observed after 5 days at 25℃ in a greenhouse. Symptoms were similar to those observed under field conditions. Control plants remained symptomless. Six isolates were reisolated from infected leaves and all confirmed to be F. avenaceum based on morphological observations and molecular identification. To our knowledge, only Septoria anthrisci has been previously reported as a pathogen of A. sinensis leaf spot (Wang et al. 2018), and this is the first report of F. avenaceum causing this disease. This discovery needs to be considered in developing and implementing disease management programs in A. sinensis production.