乳果糖-凝结芽孢杆菌合生素通过Toll样受体4/髓样分化因子88/核因子-κB信号通路缓解脂多糖诱导的断奶仔猪肠道炎症

Lactulose and Bacillus coagulans Synbiotic Alleviates Lipopolysaccharide-Induced Intestinal Inflammation in Weaned Piglets through Toll-LikeReceptor 4/Myeloid Differentiation Factor 88/NuclearFactor-κB Signaling Pathway

本试验旨在研究乳果糖-凝结芽孢杆菌合生素对脂多糖(LPS)诱导的断奶仔猪肠道炎症缓解作用的分子机制。试验选取24头健康、体重接近的27~28日龄“杜×长×大”三元杂交断奶阉公猪,随机分为4组,每组6个重复,每个重复1头猪。对照组(CON组)和LPS组饲喂基础饲粮,金霉素组(CTC组)饲喂基础饲粮+75 mg/kg的金霉素,合生素组(SYN组)饲喂基础饲粮+10 g/kg的乳果糖和2×109 CFU/kg的凝结芽孢杆菌。试验期32 d。试验结束时,对LPS组、CTC组和SYN组仔猪腹腔注射100μg/kg BW LPS,CON组注射相同剂量的生理盐水;注射4 h后屠宰,采集血清和空肠黏膜样本,测定炎症细胞因子含量及Toll样受体4(TLR4)/髓样分化因子88(MyD88)/核因子-κB(NF-κB)信号通路相关基因和蛋白表达。结果表明:1)与CON组相比,LPS刺激不仅显著提高血清促炎因子白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、白细胞介素-12(IL-12)、干扰素-γ(IFN-γ)和肿瘤坏死因子-α(TNF-α)以及抗炎因子白细胞介素-10(IL-10)含量(P<0.05),还显著提高空肠黏膜促炎因子IL-6、IL-12和IFN-γ以及抗炎因子白细胞介素-4(IL-4)、IL-10和转化生长因子-β1(TGF-β1)含量(P<0.05)。此外,与CON组相比,LPS刺激还显著提高空肠黏膜TNF-α、IL-1β与IL-10 mRNA相对表达量(P<0.05),且显著提高空肠黏膜TLR4、MyD88和NF-κB mRNA和蛋白相对表达量(P<0.05)。2)与LPS组相比,CTC组和SYN组血清IL-1β和IL-10以及空肠黏膜IL-6、IL-12、IL-4和IL-10含量显著降低(P<0.05),空肠黏膜TGF-β1含量显著提高(P<0.05),空肠黏膜TNF-α、IL-1β与IL-10 mRNA相对表达量显著降低(P<0.05),空肠黏膜NF-κB mRNA相对表达量以及TLR4和NF-κB蛋白相对表达量显著降低(P<0.05);SYN组血清IL-6、IL-8、IL-12和TNF-α以及空肠黏膜IL-1β含量显著降低(P<0.05),空肠黏膜TLR4和MyD88 mRNA相对表达量以及MyD88蛋白相对表达量显著降低(P<0.05)。综上所述,在无抗饲粮中添加乳果糖-凝结芽孢杆菌合生素替代金霉素可通过抑制断奶仔猪空肠黏膜TLR4/MyD88/NF-κB信号通路,降低TNF-α和IL-1βmRNA相对表达量,改善空肠和血清炎症细胞因子水平,从而缓解LPS诱导的应激反应,提高断奶仔猪的抗应激能力。

The aim of this study was to investigate the molecular mechanism of the alleviating effects of lactulose and Bacillus coagulans synbiotic on intestinal inflammation induced by lipopolysaccharide(LPS)in weaned piglets.A total of 24 healthy Duroc×Landrace×Yorkshire crossbred weaned boars aged of 27 to 28 days with similar body weight were randomly divided into 4 groups with 6 replicates per group and 1 pig per replicate.Piglets in the control group(CON group)and LPS group were fed a basal diet,those in the chlorotetracycline group(CTC group)were fed the basal diet+75 mg/kg chlorotetracycline,and those in the synbiotic group(SYN group)were fed the basal diet+10 g/kg lactulose and 2×109 CFU/kg Bacillus coagulans,respectively.The experiment lasted for 32 days.At the end of the experiment,piglets in LPS group,CTC group and SYN group were intraperitoneally injected with 100μg/kg BW LPS,and those in CON group were injected with the same dose of normal saline.The serum and jejunal mucosa samples were collected and the contents of inflammatory cytokines and the expression of genes and proteins related to Toll-like receptor 4(TLR4)/myeloid differentiation factor 88(MyD88)/nuclear factor-κB(NF-κB)signaling pathway were determined.The results show as follows:1)compared with CON group,LPS stimulation not only significantly increased the contents of pro-inflammatory factors such as interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-8(IL-8),interleukin-12(IL-12),interferon-γ(IFN-γ)and tumor necrosis factor-α(TNF-α)as well as the anti-inflammatory factor as interleukin-10(IL-10)in serum,but also significantly increased the contents of pro-inflammatory factors such as IL-6,IL-12 and IFN-γas well as anti-inflammatory factors such as interleukin-4(IL-4),IL-10 and transforming growth factor-β1(TGF-β1)in jejunal mucosa(P<0.05).In addition,compared with CON group,LPS stimulation also significantly increased the mRNA relative expression levels of TNF-α,IL-1βand IL-10 in jejunal mucosa(P<0.05),and significantly increased the mRNA and protein relative expression levels of TLR4,MyD88 and NF-κB in jejunal mucosa(P<0.05).2)Compared with LPS group,the contents of IL-1βand IL-10 in serum and contents of IL-6,IL-12,IL-4 and IL-10 in jejunal mucosa in CTC group and SYN group were significantly decreased(P<0.05),the TGF-β1 content in jejunal mucosa was significantly increased(P<0.05),the mRNA relative expression levels of TNF-α,IL-1βand IL-10 in jejunal mucosa were significantly decreased(P<0.05),and the mRNA relative expression level of NF-κB and the protein relative expression levels of TLR4 and NF-κB in jejunal mucosa were significantly decreased(P<0.05);the contents of IL-6,IL-8,IL-12 and TNF-αin serum and IL-1βcontent in jejunum mucosa in SYN group were significantly decreased(P<0.05),and the mRNA relative expression levels of TLR4 and MyD88 and protein relative expression level of MyD88 in jejunum mucosa were significantly decreased(P<0.05).In conclusion,dietary lactulose and Bacillus coagulans synbiotic instead of chlorotetracycline in antibiotic-free diets can inhibit the TLR4/MyD88/NF-κB signaling pathway in jejunal mucosa of weaned piglets,reduce the mRNA relative expression levels of TNF-αand IL-1β,improve the levels of inflammatory cytokines in jejunum and serum,and thus alleviate the LPS-induced stress response and improve the anti-stress ability of weaned piglets.