植物乳杆菌后生元缓解小鼠沙门氏菌感染的作用效果及机理

Effects and mechanism of Lactobacillus plantarum postbiotics on protecting against Salmonella enterica Typhimurium infection in mice

摘要:

【目的】研究不同剂量的植物乳杆菌后生元缓解小鼠伤寒沙门氏菌(Salmonella enterica Typhimurium, ST)感染的作用效果及分子机制。【方法】选择60只5周龄的C57BL/6小鼠，分为5组：Control、ST、CFS-L+ST、CFS-M+ST和CFS-H+ST，后三组分别灌胃低、中、高剂量植物乳杆菌后生元，即50、100和200 μL的无细胞培养上清(cell-free culture supernatant, CFS)预处理21 d，然后在第22天灌胃3×10⁸ CFU ST进行攻毒，3 d后采样。【结果】与对照组相比，饲喂低、中剂量CFS小鼠体重增长无明显变化，而高剂量组体重显著降低(P<0.05)。中、高剂量CFS预处理组可显著缓解沙门氏菌感染引起的小鼠体重损失(P<0.05)；饲喂3种剂量的CFS均可显著降低ST在小鼠肝脏、脾脏、结肠和脑组织的细菌移位数量(P<0.05)，并缓解结肠和脾脏病理损伤。ST攻毒显著降低盲肠乙酸和丁酸含量，而中剂量CFS预处理组具改善趋势，但差异不显著(P>0.05)。与ST组相比，预饲中剂量CFS可通过显著降低促炎因子白细胞介素-1β (interleukin-1β, IL-1β)、白细胞介素-6 (interleukin-6, IL-6)和肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)水平(P<0.05)，以及显著升高抑炎因子白细胞介素-4 (interleukin-4, IL-4)和白细胞介素-10 (interleukin-10, IL-10)水平(P<0.05)缓解炎症反应。进一步研究发现，与ST组相比，CFS-M+ST组热蛋白结构域相关蛋白3 [nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing protein 3, NLRP3]炎症小体关键基因NLRP3、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein, ASC)、半胱氨酸蛋白酶(caspase-1)和细胞死亡调节蛋白(gasdermin D, GSDMD)的mRNA表达水平显著降低(P<0.05)，其上游核因子-κB (nuclear factor kappa beta, NF-κB)通路中关键分子髓样分化因子(myeloid differentiation factor 88, MyD88)、肿瘤坏死因子受体相关因子6 (tumor necrosis factor receptor-associated factor 6, TRAF6)、转化生长因子β激活激酶1 (transforming growth factor beta-activated kinase 1, TAK1)和NF-𝜅B的mRNA表达量显著降低(P<0.05)。【结论】植物乳杆菌后生元CFS可通过抑制NF-κB通路介导的NLRP3炎症小体缓解小鼠沙门氏菌感染和炎症反应，并且预饲中剂量CFS效果最佳。

Abstract:

[Objective] To investigate the effects and mechanism of Lactobacillus plantarum postbiotics at different doses on ameliorating Salmonella enterica Typhimurium (ST) infection in mice. [Methods] Sixty 5-week C57BL/6 mice were randomized into five groups: Control, ST, CFS-L+ST, CFS-M+ST, and CFS-H+ST. Lactobacillus plantarum postbiotics (cell-free supernatant, CFS) was administrated at low (L), medium (M), and high (H) doses (50, 100, and 200 μL, respectively) for 21 days. On day 22, mice were orally challenged with ST at 3×10⁸ CFU, and the samples were collected three days later. [Results] Compared with the control group, CFS-L+ST and CFS-M+ST groups showed no significant changes in body weight gain, while the CFS-H+ST group showed a significant decrease (P<0.05). The CFS-M+ST and CFS-H+ST groups alleviated ST-induced body weight loss (P<0.05). CFS pretreatment reduced ST-induced bacterial translocation in the colon, liver, spleen, and brain (P<0.05) and alleviated the pathological damages in the colon and spleen. ST reduced the levels of acetic acid and butyric acid in the cecum, which, however, increased in the CFS-M+ST group (P>0.05). Compared with the ST group, CFS-M+ST alleviated the inflammatory response by lowering the levels of pro-inflammatory cytokines including interleukin-1beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) (P<0.05) and elevating the levels of anti-inflammatory cytokines including interleukin-4 (IL-4) and interleukin-10 (IL-10) (P<0.05). Moreover, CFS-M+ST suppressed ST-induced inflammation by modulating the nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome, as indicated by the down-regulated mRNA levels of NLRP3, apoptosis-associated speck-like protein (ASC), cysteine-dependent aspartate-specific protease 1 (caspase-1), and gasdermin D (GSDMD) (P<0.05). Furthermore, CFS inhibited NLRP3 inflammasome by blocking the upstream key nuclear factor kappa beta (NF-κB) pathway, as indicated by the down-regulated expression levels of myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), transforming growth factor beta-activated kinase 1 (TAK1), and NF-κB (P<0.05). [Conclusion] L. plantarum postbiotics CFS alleviated the S. enterica Typhimurium infection and inflammatory responses in mice by inhibiting the NF-κB-mediated NLRP3 inflammasome, and the pretreatment with medium-dose CFS showed the best effects.