松针多糖对鸡巨噬细胞HD11的天然免疫调节

doi:10.3864/j.issn.0578-1752.2020.15.017

Abstract

Abstract:

【Objective】 The innate immunomodulatary effect of pine needle polysaccharide on chicken macrophage HD11 were examined to provide theoretical ground for broiler disease prevention and treatment.【Method】Different concentrations of pine needle polysaccharide were used to treat the macrophage HD11. The experiment was divided into blank control group, positive control group (1 μg·mL^-1 was the final concentration of LPS), and pine needle polysaccharide group (25, 50, 100, 200 and 400 μg·mL^-1 were the final concentration of pine needle polysaccharide). MTT method was used to evaluate the effect of pine needle polysaccharide on the proliferation of HD11 cells. the content of NO in the supernatant of HD11 cells was determined, phagocytic function was detected by neutral red, the release of IFN-α, iNOS, IL-10, IL-6 and TNF-α were detected by ELISA, the levels of iNOS mRNA in the HD11 cells were detected by PCR.【Result】The results of MTT show, there was no significant difference among all groups (25, 50, 100, 200, 400 μg·mL^-1) in proliferation of HD11 cells. It indicated that there was no toxicity to macrophages in the range of 25-400 μg·mL^-1, and the immunoregulation experiment could be carried out. The immunomodulatory test results showed that, the concentration of NO, phagocytic function were higher significantly (P<0.01) than control group. The release of cytokines IFN-α and iNOS were higher significantly (P<0.01 or P<0.05) than control group. The amount of cytokines IL-10 was lower significantly (P<0.01 or P<0.05) than control group. The content of cytokines IL-6 and TNF-α were higher significantly (P<0.01) than control group when the pine needle polysaccharide concentration were 50, 100, 200, 400 μg·mL^-1. the concentration of NO was lower significantly (P<0.01) than positive control group. The release of cytokines IFN-α, iNOS and IL-10 were lower significantly (P<0.01 or P<0.05) than positive control group. The phagocytic function and the content of cytokines IL-6 was lower significantly (P<0.01) than positive control group when the pine needle polysaccharide concentration were 25, 50, 100 μg·mL^-1. The content of cytokines TNF-α was lower significantly (P<0.01) than positive control group when the pine needle polysaccharide concentration were 25, 50, 100, 200 μg·mL^-1.【Conclusion】 The results showed that pine needle polysaccharide can activate macrophages and enhance the innate immune function of macrophages HD11, and there was a dose-dependent effect.

Key words: needles polysaccharide, macrophage, immune regulation, cytokine

CUI XiaoZhen,LUAN Yan,LI TingTing,YANG Yu,GUAN WenChao,ZHANG Kai,WANG FuChuan,SONG XianYi. Innate Immunomodulatory Effect of Pine Needle Polysaccharide on Chicken Macrophage HD11[J].Scientia Agricultura Sinica, 2020, 53(15): 3180-3186.

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References 33

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基因名称 Primer name	引物名称 Primer name	引物序列 Primer sequence	退火温度 Annealing temperature (℃)	产物长度 Products size ( bp)
iNOS	iNOS-F	5′-GCTATTGGGCAAGCGAGAA-3′	58.9	150
iNOS	iNOS-R	5′-ATGACTTTCCCCACAGCCTTA-3′	58.9	150
GAPDH	GAPDH-F	5′- ATGCCATCACAGCCACACA -3′	58.9	127
GAPDH	GAPDH-R	5′-TGACCATCAGGGAGTTCATAGC-3′	58.9	127

组别 Group	浓度 Concentration (μg·mL^-1)	OD值 Value of OD
空白对照组 Control group	0	0.3984±0.02Bb
松针多糖组 Pine needles polysaccharide group	25	0.4318±0.01ABab
	50	0.4382±0.02ABabc
	100	0.4548±0.02ABbc
	200	0.4708±0.02Bbc
	400	0.4792±0.01Bbc

组别 Group	浓度 Concentration (μg·mL^-1)	OD值 Value of OD
空白对照组 Control group	0	21.36±0.59Aa
脂多糖组 LPS group	1	54.67±1.72Ff
松针多糖组 Pine needles polysaccharide group	25	28.37±1.06Bb
	50	31.07±0.28BCbc
	100	34.63±1.61CDcd
	200	37.84±1.77DEde
	400	41.48±1.77Ee

组别 Group	浓度 Concentration (μg·mL^-1)	中性红吞噬率 Neutral red phagocytosis (%)
空白对照组 Control group	0	100.00±2.31Aa
脂多糖组 LPS Group	1	151.72±3.18Dc
松针多糖组 Pine needles polysaccharide group	25	132.28±2.08Bb
	50	135.81±1.73Bb
	100	136.89±3.35BCb
	200	146.41±2.49CDc
	400	146.81±2.10CDc

组别 Group	浓度 Concentration (μg·mL^-1)	干扰素-α IFN-α (ng·L^-1)	白细胞介素-6 IL-6 (ng·L^-1)	白细胞介素-10 IL-10 (ng·L^-1)	一氧化氮合酶 INOS (ng·mL^-1)	肿瘤坏死因子-α TNF-α (ng·L^-1)
空白对照组 Control group	0	27.18±0.50Aa	22.68±0.88Aa	37.14±1.05Bd	0.63±0.01Aa	12.46±0.63Aa
脂多糖组 LPS group	1	67.93±1.28Df	38.53±1.20Cd	41.55±1.41Ce	1.60±0.04Ee	24.80±0.57Dd
松针多糖组 Pine needles polysaccharide group	25	31.61±0.88Ab	24.68±0.81Aa	33.28±0.98Bc	0.86±0.02Bb	12.90±0.53Aa
	50	41.47±1.20Bc	31.70±1.27Bb	26.54±1.30Ab	0.93±0.04BCbc	17.16±0.41Bb
	100	58.50±1.95Cd	32.14±1.22Bbc	25.89±1.03Aab	1.01±0.03CDc	18.08±0.53Bb
	200	59.60±1.90Cd	35.20±1.28BCbcd	25.51±0.97Aab	1.03±0.04CDc	21.61±0.75Cc
	400	63.63±1.15CDe	35.58±1.46BCcd	22.67±0.76Aa	1.14±0.03Dd	24.47±0.66Dd

Innate Immunomodulatory Effect of Pine Needle Polysaccharide on Chicken Macrophage HD11

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