白术多糖超大介孔二氧化硅纳米颗粒的制备及其黏膜免疫佐剂活性的研究

doi:10.11843/j.issn.0366-6964.2025.05.045

Abstract

Abstract:

The aim of this study was to develop a new type of traditional Chinese medicine polysaccharide mucosal immune adjuvant by combining the advantages of polysaccharide from Atractylodes macrocephala Koids and ultra-large mesoporous silica. First, ultra-large mesoporous silica nanoparticles loading polysaccharide from Atractylodes macrocephala Koidz (AMP-UCMS) was prepared and a series of characterizations were carried out on it, including transmission electron microscopy, particle size, potential, polymer dispersity index (PDI) and infrared spectroscopy. Then, the mucosal immune adjuvant activity of oral AMP-UCMS as an inactivated H9N2 avian influenza virus was further studied. In the experiment, 150 chicks were randomly divided into 6 groups and received different immunization treatments, namely negative control (Control), whole inactivated virus (WIV), WIV+AMP, WIV+UCMS, WIV+AMP-UCMS, and commercially available vaccine (Vaccine). Among them, 6 chicks from each group were intranasally challenged with H9N2 avian influenza virus. The hemagglutination inhibition (HI) test was used to determine the HI antibody titer of chicken serum in each group; the ELISA method was used to detect the specific sIgA antibody level of H9N2 avian influenza virus in the jejunum; the HE staining was used to observed the histomorphology changes of the jejunum and lung; real-time fluorescent quantitative PCR (RT-qPCR) was used to determine the mRNA expression levels of IgA J-chain, pIgR, TNF-α and IL-1β in the jejunum and various indicators such as the viral load in the lungs of chickens after challenge. The results showed that compared with the Control group, WIV+AMP-UCMS group could highly significantly increase the HI antibody titer of chicken serum (P < 0.000 1) and the specific sIgA antibody level in the jejunum (P < 0.01), and highly significantly reduce the viral load in the lungs (P < 0.000 1), and the mRNA expression levels of chicken jejunum IgA J-chain(P < 0.001), pIgR(P < 0.000 1), TNF-α and IL-1β (P < 0.000 1) were all highly significantly increased. Additionally, it could increase the number of intraepithelial lymphocytes (iIELs) in the chicken jejunum and reduce lung tissue damage following viral challenge. In conclusion, polysaccharide from Atractylodes macrocephala Koidz was successfully loaded into ultra-large mesoporous silica in the current study, and AMP-UCMS could effectively induce systemic immunity and mucosal immune responses in chickens. These research results provide a reference for the development and application of new traditional Chinese medicine polysaccharide mucosal immune adjuvants in the field of oral vaccines for livestock and poultry.

Key words: polysaccharide from Atractylodes macrocephala Koidz, ultra-large pore mesoporous silica nanoparticles, mucosal immune adjuvants, characterization, inactivated H9N2 avian influenza virus

CLC Number:

S859.797

LIN Xinyi, JIANG Xinyu, SU Zinuo, WANG Yuling, RUAN Shiyu, HONG Hailong, WU Jiahao, BO Ruonan. Study on Preparation and the Mucosal Immune Adjuvant Activity of Ultra-Large Mesoporous Silica Nanoparticles Loading Polysaccharide from Atractylodes macrocephala Koidz[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2507-2519.

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分组Group	处理方式Treatment
Control	口服0.3 mL生理盐水
WIV	口服0.3 mL WIV
WIV+AMP	口服12 mg AMP和0.3 mL WIV
WIV+UCMS	口服12 mg UCMS和0.3 mL WIV
WIV+AMP-UCMS	口服12 mg AMP-UCMS和0.3 mL WIV
Vaccine	肌注0.3 mL市售H9N2灭活疫苗

基因名称 Gene Name	引物序列(5′→3′) Primer sequences (5′→3′)
pIgR-F	TCGATGTCAGCCTGGAGGTC
pIgR-R	AGGGCATTCAATGGTCACATTTC
IgA J-chain-F	GGATCCTAATGAGGACATTGTGGAG
IgA J-chain-R	CTGGGTGGCAGTAACAACCTGA
H9N2-M-F	TGAGCGTGAACACAAACCCT
H9N2-M-R	TGAGCGTGAACACAAACCCA
IL-1β-F	ATCTCGCAGCAGCACATCA
IL-1β-R	CCAGCAGGTTATCATCATCATCC
TNF-α-F	ACTCCAGGCGGTGCCTATGT
TNF-α-R	GTGAGGGTCTGGGCCATAGAA
GADPH-F	ATGGTGAAGGTCGGTGTGAA
GADPH-R	CCTTGACTGTGCCGTTGAAT

组分 Component	体积/μL Volume
Hieff^® qPCR SYBR Green Master Mix	10
正向引物Forward Primer (10 μmol·L^－1)	0.4
反向引物Reverse Primer (10μmol·L^－1)	0.4
模板DNA Template DNA	6
无菌超纯水Ultrapure Water	3.2

程序 Process	温度 Temperature	时间 Time	循环数 Cycle number
预变性Pre-denaturation	95℃	5 min	1
变性Denaturation	95℃	10 s	40
退火、延伸Refolding, extend	60℃	30 s	40
熔解曲线Melting curve	仪器默认设置 Instrument default setting		1

样品Sample	UCMS	AMP-UCMS
粒径/nm Particle size	390.4±9.19	523.0±6.12
聚合物分散性指数Polymer dispersity index (PDI)	0.243±0.01	0.323±0.03
Zeta电位/mV Zeta potential	-8.23±0.26	-17.4±0.30

Study on Preparation and the Mucosal Immune Adjuvant Activity of Ultra-Large Mesoporous Silica Nanoparticles Loading Polysaccharide from Atractylodes macrocephala Koidz

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