In this study, the polysaccharide fraction (IOP) was isolated and purified from I. obliquus extracts, and the protective effect of IOP on dermal fibroblasts in a glycosylation environment induced by stimulation with MGO, was revealed. MGO and GO are highly reactive dicarbonyl compounds that form during glycosylation and precursor structures of CML, one of the most prevalent AGEs in the skin that is also an excellent AGE inducer (22). Studies by Guillon (23) et al. similarly showed that carbonyl compounds are excellent cellular models with which to study skin complications associated with diabetes. Therefore, in this study, 4 mM MGO was used to treat skin fibroblasts to simulate the accumulation of AGEs in human skin and examine the protective effect of IOP on skin cells in response to this stimulus.
The extract of I. obliquus exhibited the potential to be an inhibitor of AGEs (17), but it was unclear which chemical components are the main active ingredients that cause its inhibitory effect. Therefore, in this study, the extracts of I. obliquus were further extracted using different polar solvents, and the experimental results showed that the main active components contained in different extraction fractions were different. The ethyl acetate fraction contained a higher polyphenol content of approximately 12.85%, the polysaccharide fraction showed a much higher polysaccharide content of approximately 29.9%, and the n-hexane fraction contained mainly triterpene components (approximately 33.23%). The antiglycation ability of different fractions was assessed by establishing an in vitro BSA-glucose model and detecting the fluorescence intensity of the AGEs formed after incubation with BSA-glucose. The experimental results showed that the antiglycation activity of the polysaccharide fraction was higher than that of the other fractions at different concentrations; thus, the polysaccharide component may be the main active constituent of I. obliquus extract that exerts AGE inhibitory effects. The polysaccharide fraction inhibited the final formation of AGEs by trapping and reducing the production of carbonyl compounds during the glycation reaction.
Polysaccharide conformational relationships and activity mechanisms are studied by accurately analyzing the polysaccharide structure, and when polysaccharides are used as materials for biological applications, understanding their physical and chemical properties is helpful for processing requirements in the food industry. We further purified this polysaccharide fraction to produce the water-soluble polysaccharide IOP. From the molecular characterization and property analysis, the GPC assay showed that the relative molecular weight of IOP was 2.396×104 (± 6.626%), and that IOP was mainly composed of glucose, galactose, xylose, mannose, and arabinose (29.049:21.705:14.857:9.375:7.709). The results of FT-IR analysis showed that the structure has O-H and C-H stretching vibrations and contains characteristic peaks of α-glycosidic bonds, showing typical polysaccharide characteristics. The microstructure shown by AFM indicates that IOP forms polymers by the crosslinking and entangling of the sugar chains due to intermolecular forces, and the average roughness of the substance was 0.443. TGA of IOP showed that it has good thermal stability.
Cellular behavioral dysfunction can occur due to glycosylation, oxidative stress, and the overexpression of inflammatory factors, which are important causes of impaired dynamic cellular homeostasis, prolonged injury, and poor skin condition (9, 10). The experimental results showed that IOP could reduce the formation of CML (the most prevalent AGE in the skin), showing good inhibitory effects (up to 47.30%) in the range of 6–24 µg/mL.
Studies (24, 25) have shown that AGEs increase the expression level of ROS by activating NADPH oxidase, causing cells to undergo oxidative stress, which is the most important mechanism by which the inflammatory response is induced. In addition, this increase in ROS expression is an important factor in the simultaneous formation of endogenous AGEs and oxidative stress. When an organism is under prolonged oxidative stress, the body's original defense mechanisms are depleted, leading to the overproduction and accumulation of ROS, which eventually results in the production of large amounts of AGEs; thus, a feedback loop is initiated. Therefore, reducing oxidative stress caused by glycosylation and decreasing the overexpression of inflammatory factors are important means to inhibit the formation of AGEs and improve skin cell damage caused by the induction of glycosylation (26, 27). In this study, IOP significantly alleviated MGO-induced oxidative stress and effectively reduced the secretion of IL-1β, IL-6, and TNF-α, which are related inflammatory factors in cells.
In addition, AGEs tend to accumulate in the ECM of the dermis and alter the expression of ECM-related genes in fibroblasts, among which the overexpression of MMP-1, MMP-2 and MMP-9 leads to a significant decrease in the expression of fibronectin-1, COL-1 and Laminin-5, which are important components of the ECM (28–30). These proteins are important for the normal behavioral functions (including migration and adhesion) of skin cells, and among them, Laminin-5 is a major component of the cellular basement membrane that stimulates cell adhesion and motility during cell development. In a series of cascade reactions, fibroblasts eventually undergo behavioral dysfunction and cause an impaired skin condition. In this study, MGO was used to induce the glycosylation of skin fibroblasts (CCC-ESF-1 cells), and the experimental results showed that IOP could reduce cell death after stimulation with different concentrations of MGO. The changes in MMPs 1, 2 and 9, FN-1, LM-5 and COL-1 mRNA expression in cells were measured by qRT‒PCR. The experimental results showed that IOP could significantly alleviate the MGO-induced increase in MMPs 1, 2 and 9 mRNA expression, followed by upregulation of ECM protein (FN-1, COL-1 and Laminin-5) mRNA expression. The results of the cell scratching and adhesion assays showed that IOP effectively restored the migration and adhesion functions and improved the MGO-induced behavioral dysfunction of fibroblasts.
As shown in Fig. 8, the above results suggest that IOP can effectively inhibit MGO-induced skin glycosylation reactions and ameliorate glycosylation-induced skin cell damage, such as oxidative stress, inflammation, and behavioral dysfunction, showing its potential application as an AGE inhibitor in the field of skin care.