Polysaccharide from Atractylodes macrocephala Koidz binding with zinc oxide nanoparticles: Characterization, immunological effect and mechanism

Abstract

Atractylodes macrocephala Koidz (A. macrocephala) has been used both as a traditional medicine and functional food for hundreds of years in Asia. And it has a variety of biological activities, such as enhancing the ability of immunity and modulating effect on gastrointestinal motility. In this study, a watersoluble polysaccharide with molecular weight of 2.743 × 10 3 Da was isolated from the root of A. macrocephala. Polysaccharide from A. macrocephala (AMP) consisted of arabinose, galactose, glucose, xylose, mannose, ribose, galactose uronic acid, glucose uronic acid, with a percentage ratio of 21.86, 12.28, 34.19, 0.43, 0.92, 0.85, 28.79, and 0.67%, respectively. Zinc plays an important role in immune system. Therefore, we supposed that AMP binding with zinc oxide (ZnO) nanoparticles (AMP-ZnONPs) might be an effective immunostimulator. AMP-ZnONPs was prepared by Borch reduction, and its structural features were characterized by Scanning Electron Microscope (SEM), Transmission electron microscope (TEM), TEM-energy dispersive spectroscopy mapping (TEM-EDS mapping), Fourier transform infrared spectroscopy (FT-IR), X-ray photoelectron spectrometer (XPS), X-ray diffraction (XRD), particle size and zeta-potential distribution analysis. Then, its immunostimulatory activity and the underlying mechanism were evaluated using RAW264.7 cells. The results showed that AMP-ZnONPs remarkably promoted cell proliferation, enhanced phagocytosis, the release of nitric oxide (NO), cytokines (IL-6 and IL-1β) and the expression of co-stimulatory molecules (CD80, CD86 and MHCII). Moreover, AMP-ZnONPs could promote Frontiers in Nutrition 01 frontiersin.org Bo et al. 10.3389/fnut.2022.992502the expression of Toll-like receptor 4 (TLR4), Myeloid differentiation factor 88 (MyD88), TNF receptor associated factor 6 (TRAF6), phospho-IκBα (P-IκBα) and phospho-p65 (P-p65), and TLR4 inhibitor (TAK242) inhibited the expression of these proteins induced by AMP-ZnONPs. Therefore, AMP-ZnONPs activated macrophages by TLR4/MyD88/NF-κB signaling pathway, indicating that AMP-ZnONPs could act as a potential immunostimulator in medicine and functional food. KEYWORDSpolysaccharide from Atractylodes macrocephala Koidz, zinc oxide nanoparticles, immunostimulatory activity, TLR4 signaling pathways, potential immunostimulator Materials and methods Reagents and materialsA. macrocephala was purchased from the Tongrentang Company in Beijing. The plant material was identified by Prof. Jingui Li. The purified AMP was prepared in our laboratory and the polysaccharide content was 96% (UV). RAW264.7 cells were obtained from American Type Culture Collection (ATCC,

Mucosal immune responses and protective efficacy elicited by oral administration AMP-ZnONPs-adjuvanted inactivated H9N2 virus in chickens

“…AMP-ZnONPs was prepared according to the protocol described in our previous study ( Bo et al, 2022 ). Chicken immunoglobulin G ( IgG ) ELISA kit was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.…”

“…Recent studies have revealed the potential of polysaccharides as immune adjuvants, enhancing both specific and non-specific immune responses when combined with vaccines ( Smith et al, 2023 ; Zhao et al, 2023 ). Polysaccharide from Atractylodes macrocephala Koidz binding with zinc oxide nanoparticles ( AMP-ZnONPs ), a novel compound obtained through reductive amination reactions in our lab, have shown potential in activating the immune functions of RAW264.7 cells and serving as vaccine adjuvants ( Bo et al, 2022 ).…”

Polysaccharide from Atractylodes macrocephala Koidz Binding with Zinc Oxide Nanoparticles as a Novel Mucosal Immune Adjuvant for H9N2 Inactivated Vaccine

“…The AMP-ZnONPs were prepared via the Borch reaction as previously described [14]. To prepare the H9N2 vaccine formulation, 1 mg/mouse of ZnONPs, 4 mg/mouse of AMP, 4 mg/mouse of AMP-ZnONPs and 5 µg/mouse of CT were mixed separately with 0.2 mL 10 −8.83 EID 50 /0.1 mL/dose of WIV, and the mixture was incubated for 24 h at 4 • C 10 rpm on a rotary mixer (MX-RD-Pro, DLAB, Beijing, China).…”

“…RNA extraction, cDNA synthesis and qRT-PCR were performed on samples of the jejunum as previously described [14]. Primer sequences GAPDH, α-chain, J-chain, polymeric immunoglobulin receptor (pIgR), IL-6, TNF-α, B-cell activating factor (BAFF), proliferation-inducing ligand (APRIL), CCR9 and CCL25 are listed in Table 3.…”

“…Previous research has explored the binding of AMP to ZnONPs (AMP-ZnONPs) and its potential application as a vaccine adjuvant. This complex was demonstrated to activate macrophages through the TLR4/MyD88/NF-κB signaling pathway [14], thereby enhancing immunity.…”