Engineering Success

The purpose of this study is to study the affinity of six main chemicals in rose flavonoids with GRIN2B protein by molecular docking technique (Agu et al., 2023 ). We divided the project into four modules: cell damage model building module, protein structure modeling module, small molecule preparation module and docking analysis module. We screened out the specific gene GRIN2B through transcriptomics detection and data analysis, modeled it with the target protein GRIN2B of AlphaFold3, and optimized the structure of the main chemical substances in flavonoids from protein and Rose by Maestro calculation software. The predicted protein structure and small molecules were optimized to ensure stability and minimize energy, which significantly increased the possibility and accuracy of successful docking. Then, according to the semi-flexible docking method, the optimal binding mode and reasonable molecular binding pocket are analyzed. We expect that the six main chemicals in rose flavonoids will closely bind with GRIN2B protein, which will help us understand their effects of relieving inflammation and oxidative damage.

1.Fresh rose was purchased, and we selected 100 g petals. After washing with sterile water and drying in oven, the dried petals (about 5 g) were crushed. Accurately 1.0g of rose petal powder was weighed, which was extracted with distilled water at a ratio of 1:10 (i.e. 10 mL) in a water bath at 65 ℃ for 2 hours. The extraction was repeated for three times (for a total of 6 hours), then the extraction solution was centrifuged for 15 min (1500 rpm), and the supernatant (for a total of 20 mL) was combined together. The total amount of flavonoids in the supernatant is the total content of flavonoids in rose petals. (Wu et al., 2022)

2.Two types of cells (PC12 cells and N2a cells) were cultured in the DMEM medium containing 10% FBS and 1% penicillin/streptomycin, in a humidified incubator at 37 ℃ with 5% CO2. The growth medium was replaced everyday and the cells were propagated every three days. For cells injury model construction, Aβ protein 42 (10 μM) was added into the cells and co-cultured for 24 hours. Then, several inflammatory factors including NF-κB, TNF-α and IL-1β were measured to confirm the successful model construction.

3.For GRIN2B is membrane protein, we can choose mammalian or insect cell expression system, and the plasmid is designed as follows. By cloning into a vector, expression plasmids encoding functional proteins are further generated and transformed into DH5α-competent cells. Then, plasmids are extracted and constructed from positive clones and confirmed by sequencing. Next, extract the protein from the plasmid for purification. (Li et al., 2022, Li et al., 2024)

4.Introduce the overexpression vector into expression-deficient cells for the purpose of interaction validation or to apply stress treatment to the overexpressing cells.

1.To screen out the special sensors (genes) of rose flavonoids extraction in PC12 cells, which was under Aβ protein 42 (10 μM) treatment, we gathered four groups of cells, control without treatment (C), model (M), control+rose flavonoids extraction (C+D), model+rose flavonoids extraction (M+D), and performed transcriptomics detection and data analysis.

2.To quantify protein expressions of GRIN2B, associated proteins NF-κB, TNF-α and IL-1β, with respect to the control protein β-actin, WB is performed. The relative amounts of proteins in each group were quantified concerning β-actin expression. characterized by the up-regulation of GRIN2B, alongside the down-regulation of NF-κB, TNF-αand IL-1β.

3.Conduct morphological, protein, and molecular level assessments on cells successfully transfected with the plasmid to confirm the successful construction of the vector and the functionality of the protein pathway

1.Through experiments, we verified the binding model and different affinities between six main chemical substances in rose flavonoids (Kaempferol, Quercetin, Juglanin, Avicularin, Astragalin, Hyperoside) and GRIN2B protein. Rose flavonoids in this pathway can effectively alleviate the risk of Alzheimer' s disease caused by circadian rhythm disorder.

2.We found that GRIN2B may be a sensitive factor to alleviate inflammation and oxidative damage of PC12 cells, and rose flavonoids can up-regulate PC12 cells. Expression level of GRIN2B in cells. Based on these characteristics, we prepared a functional tea bag of rose flavonoids.

1.Agu P C , Afiukwa C A , Orji O U ,et al.Molecular docking as a tool for the discovery of molecular targets of nutraceuticals in diseases management[J].Scientific Reports, 2023, 13(1).DOI:10.1038/s41598-023-40160-2.
2.Wu Mengqi, et al."Purification and Identification of Flavonoid Molecules from Rosa setate x Rosa rugosa Waste Extracts and Evaluation of Antioxidant, Antiproliferative and Antimicrobial Activities."Molecules 27.14(2022):4379-4379.
3.Li, Y., Liu Y., Huang, M., &Chen, M. (2022). Batch construction and screening recombinant plasmid by connecting DNA solution with plasmid vector. CN113674804A
4.Weiyi Li,Darach Miller,Xianan Liu,Lorenzo Tosi,Lamia Chkaiban,Han Mei... & Sasha F Levy.(2024).Arrayed in vivo barcoding for multiplexed sequence verification of plasmid DNA and demultiplexing of pooled libraries..Nucleic acids research