Black mulberry (Morus nigra L.) polysaccharide ameliorates palmitate-induced lipotoxicity in hepatocytes by activating Nrf2 signaling pathway
Introduction
Palmitic acid (PA) is a kind of saturated fatty acid widely existing in animal cell and adipose tissue, and it is also the main component of refined palm oil [1,2]. Generally, it is obtained from diet or synthesized endogenously through de novo synthetic reaction in human body [3,4]. However, increasing evidence indicates that PA-induced lipotoxicity is closely related to the pathological processes of oxidative stress, ROS overproduction and hepatocytes dysfunction, which also resulted in metabolism disorder and many diseases including obesity, diabetes, cardiovascular diseases and cancer [[5], [6], [7]]. At present, although some clinical drugs could promote lipid oxidation, mitigate ectopic fat accumulation, prevent or delay the damage caused by lipotoxicity, their potential side effects could not be neglected. This limits the long-term use of clinical treatment. Therefore, it is urgent to develop a natural, safe and effective agent to prevent lipotoxicity-associated metabolic disorders.
Polysaccharide, a kind of high molecular weight polymer, is derived from natural products and consists of many kinds of monosaccharides through glycosidic bonds [8]. In recent years, polysaccharides isolated and purified from healthy food, such as Ginseng, Hirsutella sinensis, Zizyphus Jujuba, and Chinese wolfberry have received increasing attentions [[9], [10], [11], [12]]. Moreover, epidemiological data have shown that plant polysaccharides have beneficial biological effects to ameliorate inflammation and oxidative stress, especially the cytotoxicity [[13], [14], [15]]. Xiong C et al. reported that MIP, a crude polysaccharide extracted from Morchella importuna, prevented PC12 cells from H2O2-induced cytotoxicity by reducing oxidative stress [16]. Kim K et al. found that aloe polysaccharides (APs) have a protective effect on free fatty acid-induced lipotoxicity in pancreatic β-cells [17]. In addition, Rubus chingii Hu-derived polysaccharides ameliorated palmitic acid-induced lipotoxicity in HepG2 cells [18]. Morus nigra L. (black mulberry), as a functional food with high edible and medicinal value, is often used in immune regulation, anti-inflammation and other purposes [19]. However, it remains unclear whether the black mulberry polysaccharide has a protective effect against hepatic lipotoxicity.
In this study, three different polysaccharides fractions were extracted from black mulberry by graded precipitation with 30%, 60% and 90% ethanol and analyzed by HPLC, FT-IR, GPC methods. Subsequently, the protective effect of black mulberry polysaccharides on PA-induced lipotoxicity in HepG2 cells and its potential molecular mechanism were investigated. In accordance with the obtained research results, black mulberry polysaccharide plays an important role in maintaining the normal redox status of hepatocytes and alleviating the disorder of lipid metabolism. Therefore, black mulberry polysaccharide has a potential ability to be used as a safe and efficient natural detoxification agent for PA-induced lipotoxicity.
Section snippets
Chemicals
RPMI-1640 medium and fetal bovine serum (FBS) were obtained from GIBCO (Grand Island, NY, USA). Glucose, rhamnose, arabinose, galactose, fucose, mannose, xylose, galacturonic acid, and glucuronic acid standard samples were purchased from Yuanye Biotechnology (Shanghai, China). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-Htetrazolium bromide (MTT), palmitic acid (PA), dihydroethidium (DHE), 2′,7′-dichlorofluorescin diacetate (DCFH-DA) and dimethyl sulphoxide (DMSO) were obtained from Sigma (St.
Yields and characterization of black mulberry polysaccharides
Different extraction methods were compared for their polysaccharide yields. The extraction yields and physicochemical properties of black mulberry fruit extracted by stepwise precipitation with 30%, 60%, 90% ethanol were summarized. As shown in Table. 1, the extraction yields of three different polysaccharide fractions were 0.53 g for BP1, 0.27 g for BP2 and 0.26 g for BP3, respectively, from 100 g black mulberry fruit freeze-dried powder. Generally, BPs contained high levels of polysaccharide
Discussion
PA is the most abundant free fatty acid in human tissues [37]. PA-induced lipotoxicity in hepatocytes is closely related to the occurrence and development of inflammation, metabolic disorder, and cancer [38]. Increasing studies have shown that the elimination of ROS or the reduction of oxidation level could prevent lipotoxicity [39]. Of note, whether black mulberry polysaccharides has a protective effect against PA-induced lipotoxicity is not clear. In the present study, we found that black
Abbreviations
- BPs
black mulberry fruit polysaccharides
- BP1
black mulberry fruit polysaccharide-1
- BP2
black mulberry fruit polysaccharide-2
- BP3
black mulberry fruit polysaccharide-3
- PA
palmitic acid
- ROS
reactive oxygen species
- RH123
rhodamine 123
- GSH
glutathione
- SOD
superoxide dismutase
- CAT
catalase
- GPX
glutathione peroxidase
- Nrf2
Nuclear factor erythroid 2 related factor 2
- HO-1
heme oxygenase-1
- NQO1
NAD(P)H: quinone oxidoreductase 1
- ARE
antioxidant response element
- Keap1
kelch-like ECH-associated protein 1
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgments
This work was supported by Grants from National Natural Science Foundation of China (U1703105), Lishui Science and Technology Bureau Research Fund (No.2020zdhz01), Zhejiang Provincial Natural Science Foundation of China (LR18C200002).
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