Glucose-rich polysaccharide from dried ‘Shixia’ longan activates macrophages through Ca2+ and CR3- mediated MAPKs and PI3K-AKT pathways

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Highlights

  • An immune active polysaccharide from dried Shixia longan (LPsx) was studied.

  • LPsx was a 4.1 kDa polysaccharide composed of (1 → 6)-α-d-Glc and (1 → 6)-β-d-Glc.

  • LPsx shows immune activity via Ca2+ and CR3-mediated PI3K/AKT and MAPKs pathways.

  • LPsx also exhibits anti-inflammatory activity by regulating MAPKs and NF-κB pathways.

  • These results help for elucidating the health promoting mechanism of dried longan.

Abstract

A water-soluble glucose-rich polysaccharide from dried ‘Shixia’ longan pulp (LPsx) has been isolated for the first time, and its structure and immuno-regulatory mechanism were studied. LPsx is a hetero-polysaccharide with the average molecular weight 4102 g/mol. It was mainly consisted of glucose (95.9%), and small proportions of arabinose (2.1%), galactose (1.0%), mannose (0.6%), and xylose (0.4%). As analyzed by NMR, LPsx was mainly composed of (1 → 6)-α-d-glucose and (1 → 6)-β-d-glucose, branched with α-d-glucose-(1→. The immunomodulatory activity study showed that LPsx significantly increased the phagocytosis of macrophages, and strongly promoted the production of NO, IL-1β, IL-6 and TNF-α. Moreover, LPsx could inhibit the inflammatory response induced by lipopolysaccharide. The immuno-regulatory mechanism of LPsx was studied using RNA- sequencing and receptors activity analyses. It was found that LPsx induced macrophage activation via Ca2+ and CR3-mediated MAPKs and PI3K-AKT signaling pathways. The results would be helpful for revealing the health promoting mechanism of dried ‘Shixia’ longan in traditional Chinese medicine.

Introduction

Longan (Dimocarpus longan Lour.) is well known as a ‘medicinal and edible’ fruit, which is widely cultivated in China and southeastern Asia [1]. In China, ‘Shixia’ and ‘Chuliang’ are the most widely cultivated longan varieties. Ordinarily, ‘Chuliang’ longan is big in size and juicy in texture, while ‘Shixia’ longan is smaller and sweeter. They are commonly consumed after drying as a traditional Chinese medicine for improving body immunity and treating diseases such as neurasthenia, hypomnesia and anemia [2,3].

The health promotion function of longan is well known; however, the underneath mechanism is still poorly understood. Various bioactive compounds such as polysaccharides, corilagin, flavone glycosides, γ-aminobutyric acid, etc., have been identified in longan fruits [4]. Among these compounds, polysaccharides are the most abundant ones, which have been proposed to be a predominant component attributing to the health promotion function of longan [5,6]. Polysaccharides from natural sources have been reported to have multiple biological activities such as immune regulation, antitumor, antioxidant and so on [4,[7], [8], [9], [10]], and their bioactivities are closely associated with the sources and structural characteristics [11].

In order to fully understand the health promotion function of dried longan fruits, attentions have been paid on the structure–function relationship and bioactive mechanism of their polysaccharides. To date, immune activity of polysaccharides in ‘Chuliang’ longan fruits have been relatively well studied. It has been reported that ‘Chuliang’ longan polysaccharides are mainly composed of glucose (45–70%, 1 → 4/6-Glc), mannose (1 → 6-Man), galactose (1 → 6-Gal) and arabinose (1 → 5-Ara), and could activate macrophages through Toll-like receptor (TLR2/TLR4)-mediated MAPK and NF-κB pathways [6,12]. However, these could not elucidate the health promotion function of dried longan, because the structure and immune activity of polysaccharides vary with variety [[13], [14], [15]]. As another major longan cultivar, ‘Shixia’ has rarely been studied, with the structure and bioactivity of its polysaccharides being largely unknown. Zhu et al. [5] identified a glucose-rich polysaccharide (108 kDa) with anticancer activity in fresh ‘Shixia’ longan; however, its immune activity was not studied. In addition, it has been reported that process procedures of fruits, for instance drying, could change the structure and bioactivity of polysaccharides [[16], [17], [18]]. Hence, it was proposed that the structure and bioactive mechanism of polysaccharides in dried ‘Shixia’ longan might be significantly different from those in fresh fruits, as well as with other longan cultivars. In order to fully understand the immunity improving mechanism of dried longan as a traditional Chinese medicine, it is of importance to investigate bioactive polysaccharides in dried ‘Shixia’ longan.

The current study focused on the structure and immunoregulatory mechanism of the polysaccharide from dried ‘Shixia’ longan. Major water-soluble polysaccharides were isolated, and fine structural features including molecular weight, monosaccharide composition, functional groups, and the types of glycosidic bonds were characterized. The immunomodulatory activity was then evaluated with phagocytosis and cytokine production of RAW 264.7 macrophages, and the immune regulatory mechanism was studied with RNA-seq and membrane receptors activity. The results of this study would be beneficial for understanding the underneath mechanisms of the medicinal value especially immune regulation function of dried ‘Shixia’ longan fruits in traditional Chinese medicine.

Section snippets

Materials

Longan (Dimocarpus longan Lour. cultivars ‘Shixia’) fruits without diseases were obtained from a local orchard (Conghua, Guangdong province, China) in August 2016. After removing branches and leaves, the fresh longan fruits were dried using heat-pump at 65 °C for 48 h (the final moisture content was about 17%), and then store at 4 °C.

Dextran standards and lipopolysaccharide (LPS) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). The reagents for cell culture including DEME medium,

Molecular weight and monosaccharide composition

The molecular weight of LPsx was analyzed using HPGPC. As shown in Fig. 1A, LPsx showed a single sharp and symmetrical peak appeared at 49.77 min, and its molecular weight was 4102 g/mol. The poly-dispersion coefficient (Mw/Mn) of LPsx was 1.10, confirming that LPsx is a homogeneous polysaccharide.

The monosaccharide composition of LPsx was analysis by GC–MS (Fig. 1B), the result indicated that LPsx was mainly composed of glucose, accounting for 95.9%. Apart from glucose, it also had minor

Conclusions

A novel polysaccharide from dried ‘Shixia’ longan fruits was studied, and its structural characteristics and immune active mechanism were elucidated. It was found that LPsx is a glucose-rich polysaccharide with an average molecular weight of 4102 Da. It was composed of α-d-(1 → 6)-Glc and β-d-(1 → 6)-Glc, and branched with α-d-Glcp-(1→. LPsx could strongly enhance macrophages activity by promoting phagocytosis and improving the production of NO, IL-1β, IL-6 and TNF-α. The immunostimulant

Declaration of competing interest

The authors declare no conflict of interest.

Acknowledgments

This work was supported by the Earmarked Fund for China Agriculture Research System (No. CARS-32), the Yang Fan Innovative and Entrepreneurial Research Team Project (No. 2014YT02H013) and the International Training for Outstanding Scientific Talents Program from South China Agricultural University (No. 2018YQGP-BS007).

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