Identification of Erzhu Jiedu Recipe and its molecular mechanism underlying inhibited human hepatoma cells by UHPLC-Q-Exactive Orbitrap HRMS and network pharmacology.

ETHNOPHARMACOLOGICAL RELEVANCE: Erzhu Jiedu Recipe (EZJDR) is a formula of traditional Chinese medicine (TCM) for treating hepatitis B virus-related hepatocellular carcinoma (HBV-HCC). However, its effective components and the mechanism of action remain unclear. AIM OF THE STUDY: To explain how the active compounds of EZJDR suppress the growth of hepatoma cells. METHODS: UHPLC-Q-Exactive Orbitrap HRMS was used to identify the chemical constituents of EZJDR and their distribution in the serum and liver of mice. Together with experimental investigations, network pharmacology unraveled the molecular mechanism of components of EZJDR underlying the inhibited Hep3B cells. RESULTS: A total of 138 compounds which can be divided into 18 kinds of components (such as sesquiterpenoids, diterpenoids, anthraquinones, flavonoids and so on) were found in the aqueous extract of EZJDR. Of these components, the tricyclic-diterpenoids exhibited a highest exposure in the serum (74.5%) and liver (94.7%) of mice. The network pharmacology revealed that multiple components of EZJDR interacted with key node genes involved in apoptosis, proliferation, migration and metabolism through various signaling pathways, including ligand binding and protein phosphorylation. In vitro experiments demonstrated that 6 tricyclic-diterpenoids, 2 anthraquinones and 1 flavonoid inhibited the viability of Hep3B cells, with IC50 values ranging from 3.81 µM to 37.72 µM. Dihydrotanshinone I had the most potent bioactivity, arresting the S phase of cell cycle and inducing apoptosis. This compound changed the expression of proteins, including Bad, Bax, Bcl-2, Bal-x, caspase3 and catalase, which were associated with mitochondria-mediated apoptotic pathways. Moreover, dihydrotanshinone I increased the levels of p21 proteins, but decreased the phosphorylated p53, suggesting accumulation of p53 protein prevented cell cycle progression of Hep3B cells with damaged DNA. CONCLUSIONS: These results suggested that multiple components of EZJDR-diterpenoid, anthraquinone and flavonoid-could be the effective material for the treatment of HBV-HCC. This research provided valuable insights into the molecular mechanism of action underlying the therapeutic effects of EZJDR.

Genome-wide association study reveals a patatin-like lipase relating to the reduction of seed oil content in Brassica napus.

BACKGROUND: Rapeseed (Brassica napus L.) is an important oil crop world-widely cultivated, and seed oil content (SOC) is one of the most important traits for rapeseed. To increase SOC, many efforts for promoting the function of genes on lipid biosynthesis pathway have been previously made. However, seed oil formation is a dynamic balance between lipid synthesis and breakdown. It is, therefore, also reasonable to weaken or eliminate the function of genes involved in lipid degradation for a higher final SOC. RESULTS: We applied a genome-wide association study (GWAS) on SOC in a collection of 290 core germplasm accessions. A total of 2,705,480 high-quality SNPs were used in the GWAS, and we identified BnaC07g30920D, a patatin-like lipase (PTL) gene, that was associated with SOC. In particular, six single-nucleotide-polymorphisms (SNPs) in the promoter region of BnaC07g30920D were associated with the significant reduction of SOC, leading to a 4.7-6.2% reduction of SOCs. We performed in silico analysis to show a total of 40 PTLs, which were divided into four clades, evenly distributed on the A and C subgenomes of Brassica napus. RNA-seq analysis unveiled that BnPTLs were preferentially expressed in reproductive tissues especially maturing seeds. CONCLUSIONS: We identified BnaC07g30920D, a BnPTL gene, that was associated with SOC using GWAS and performed in silico analysis of 40 PTLs in Brassica napus. The results enrich our knowledge about the SOC formation in rapeseed and facilitate the future study in functional characterization of BnPTL genes.

Elevating seed oil content in a polyploid crop by induced mutations in SEED FATTY ACID REDUCER genes.

Plant-based oils are valuable agricultural products, and seed oil content (SOC) is the major yield component in oil crops. Increasing SOC has been successfully targeted through the selection and genetic modification of oil biosynthesis. The SOC in rapeseed declined during the seed maturation and eventually caused the final accumulated seed oil quantity. However, genes involved in oil degradation during seed maturity are not deeply studied so far. We performed a candidate gene association study using a worldwide collection of rapeseed germplasm. We identified SEED FATTY ACID REDUCER (SFAR) genes, which had a significant effect on SOC and fatty acid (FA) composition. SFAR genes belong to the GDSL lipases, and GDSL lipases have a broad range of functions in plants. After quantification of gene expression using RNA-seq and quantitative PCR, we used targeted (CRISPR-Cas mediated) and random (chemical) mutagenesis to modify turnover rates of seed oil in winter rapeseed. For the first time, we demonstrate significant increase of SOC in a crop after knocking out members of the BnSFAR4 and BnSFAR5 gene families without pleiotropic effects on seed germination, vigour and oil mobilization. Our results offer new perspectives for improving oil yield by targeted mutagenesis.