In this doctoral thesis, we first optimized the extraction method of intracellular metabolites to provide technical support for subsequent metabonomics research. Then, by creating cell wound model and UVB induced cell photoaging model, the effect of nitric oxide (NO) on in vitro wound healing model, the in vitro antioxidant capacity of maple leaves extract and the inhibition of UVB induced cell photoaging of maple leaves extract were analyzed by means of biochemistry and metabonomics. The abstract is divided into four parts, corresponding to the four topics of the present study.
Part 1
Metabolomics is a technology to analyze what is happening in living organisms, which is very useful for analyzing the current state of living organisms. In metabolome research, the complete extraction and identification of metabolites is the top priority, which is very important for comprehensive analysis of living organisms. In this study, we explored the optimal extraction protocol for metabolites in adherent cells, successively focusing on extractant, quenching and harvesting methods, and additional cell lysis. Our results shown that using a four-phase extractant (MeOH: MTBE: TCM: ddH2O = 1: 0.5: 0.5: 0.1), liquid nitrogen quenching and cell scraper harvesting of cells are optimal for the extraction of intracellular metabolites. At the same time, we applied this extraction protocol to the analysis of photoaging metabolites in HaCaT and B16 cells. Finally, we found that there were four metabolites with the same trend in both cells, namely, pyroglutamic acid and proline, which were significantly increased after UVB radiation, and lactic acid and succinic acid, which were significantly decreased. These results demonstrated that our metabolite extraction protocol can be used in future research.
Part 2
Skin trauma is a focus problem in clinical medicine, which often brings heavy psychological burden and physical obstacles to patients. In the previous studies, it was shown that exogenous supply of NO can promote wound healing. In this study, the fermentation broth of lettuce containing NO was used as the NO carrier. In order to explore the role and mechanism of fermentation broth containing NO in wound healing, this study used HaCaT to build a wound model, and used different doses of NO to treat the wound model. The role of NO in promoting wound healing was analyzed through cell activity, cell cycle, Matrix metalloproteinases (MMPs), oxidative stress level, cell migration experiment, and changes in metabolites. Our results shown that lettuce fermentation broth containing 100 nM dose of NO can improve cell activity, increase the number of cells in G1 phase, thereby promoting cell division, increase the content of MMP-2 and MMP-9, increase the rate of cell migration, and thus accelerate the rate of wound healing. After analyzing the metabolites of HaCaT cells stimulated by NO, we found that the contents of energy substances (carbohydrates), vitamins (L-ascorbic acid and dehydroascorbic acid dimer), and bioactive organic acids (glyceric acid, quinic acid, 4-aminobutyric acid) that can promote wound healing were significantly increased. In conclusion, the fermentation liquid of lettuce containing NO can accelerate wound healing by improving cell activity, shortening cell cycle, accelerating cell migration rate, and promoting cell nutrient supply.
Part 3
Acer pseudoplatanus (maple) is a widely grown ornamental plant. In addition to its ornamental and ecological value, it also has potentially high economic value. It is a rich source of polyphenols and exhibits antioxidant activity. However, the relationship between polyphenol content and antioxidant activity in maple leaves of different colors (green, yellow, and red) has not yet been investigated. In this study, the total polyphenol (TP), total flavonoid (TFlav), tannin (TET), chlorophyll a and b (Chl a and b), total anthocyanin (TAN), and total carotene (TAC) contents in maple leaves of different colors were evaluated. Their antioxidant activities were determined based on the inhibition of lipid oxidation, DPPH scavenging, ferric ion-reducing antioxidant power, and iron-chelating abilities. The concentrations of TP, TET, TFlav, TAN, and TAC in red maple leaves were higher than those in green and yellow maple leaves. In addition, red maple leaves shown a higher antioxidant effect than the leaves of the other two colors. We observed that antioxidant activity was positively correlated with TP, TFlav, and TAN and negatively correlated with Chl a and b. Finally, we analyzed the metabolites of the different colored (i.e., green, yellow, and red) maple leaves using gas chromatography/mass spectrometry (GC/MS) and found that the metabolite profile significantly varied between the different colors. These results suggest that red leaves are a good source of polyphenols and antioxidants and have potential use in the development of functional foods and medicinal applications.
Part 4
The objective of the current study was to assess the antioxidant activity of different colors of maple leaves extract (MLE) and their anti-photoaging effects on HaCaT. In this study, the photoaging cell model was prepared by irradiating HaCaT with 50mj/cm2 UVB dose. Our results show that MLE can improve cell activity and reduce UVB induced oxidative stress. In order to explore the mechanism of MLE reducing UVB induced injury, this study used GC/MS technology to analyze intracellular metabolites, and PCA and OPLSDA technology to analyze metabolites. Finally, 27 differential metabolites were identified, including carbohydrates, amino acids, organic acids. Through further analysis of metabolites, the results shown that the anti-photoaging ability was significantly improved by the increase of intracellular energy compounds (glucose, sucrose, fructose), and moisturizing compounds (lactic acid and urea). The results of this study shown that MLE has the effect of resisting cell oxidative stress, wrinkles and dryness caused by skin photoaging, and can play a role in cosmetics or functional food.